https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/api.php?action=feedcontributions&user=Joerdensv&feedformat=atomBioinformatikpedia - User contributions [en]2024-03-29T10:17:18ZUser contributionsMediaWiki 1.31.16https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28603Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T20:26:29Z<p>Joerdensv: /* Pymol analysis of average and bfactor */</p>
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<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
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<br style="clear:both;"><br />
<br />
== Short task description ==<br />
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<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
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Continuation of [[Molecular_Dynamics_Simulations_Hemochromatosis|task 8]]. Analysis of the MD simulations for the wildtype and two mutants (R224W and C282S).<br />
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<br style="clear:both;"><br />
<br />
== Protocol ==<br />
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<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
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<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells| Minimum distance between periodic boundary cells]].<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
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R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
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gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
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gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
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<br style="clear:both;"><br />
<br />
=== Energies ===<br />
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<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
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<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
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<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
Also worth noting is, that the calculation of b-factors through NMA resulted in ones (cf. <xr id="tab:avg_bfactor"/> and [[Normal_Mode_Analysis_Hemochromatosis#Atomic_fluctuations| WebNMA fluctuations]]) similar to our MD calculated wildtype b-factors. It might also be possible that Elnemo (cf. [[Normal_Mode_Analysis_Hemochromatosis#B-factors| elNemo b-factors]]) has also calculated b-factors similar to the MD ones but are hidden in this depiction by our threshold.<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (see <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the values of the following timesteps) lower at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmful mutation of the two based on the MD results, yet it is known to be one of the more malicious ones. Overall we have gained some insight into the behavior of the three variants during the MD simulations, though it was sometimes hard to make sense of the information or to decide if it might be good or bad for the protein's function.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Sequence_Alignments_Hemochromatosis&diff=28583Sequence Alignments Hemochromatosis2012-08-31T20:05:07Z<p>Joerdensv: /* Multiple Sequence Alignments */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Sequence Alignments Hemochromatosis|Task 2: Sequence alignments]]<br />
<br />
== Short Task Description ==<br />
<br />
* Apply and compare different sequence search algorithms.<br />
* Use the results for MSAs<br />
<br />
Detailed description: [[Task_alignments]]<br />
<br />
== Protocol ==<br />
<br />
We've made a protocol with the commands and scripts we used to create our results: [[Task2 Hemochromatosis Protocol | Protocol]]<br />
<br />
== Reference Sequence ==<br />
Sequence from Uniprot: [http://www.uniprot.org/uniprot/Q30201.fasta Q30201]<br />
<br />
>sp|Q30201|HFE_HUMAN Hereditary hemochromatosis protein OS=Homo sapiens GN=HFE PE=1 SV=1<br />
MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVF<br />
YDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQV<br />
ILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNR<br />
AYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWL<br />
KDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPS<br />
PSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAERE<br />
<br />
== Sequence Searches ==<br />
<br />
In this task we've tested three different sequence search algorithms against each other. We also used three different databases:<br />
* Big80 for BLAST and PSI-BLAST<br />
* Big for PSI-BLAST<br />
* Uniprot20 for HHblits<br />
<br />
The results for each search run were filtered for unique hits and an e-Value cutoff of 2e-3. The cutoff was chosen to be in accordance with the given minimum cutoff (2-e3) for the PSI-BLAST profiles. For the evaluation of the hits we used two methods:<br />
* GO term analysis: We compared each hit's GO term with the GO terms of HFE (Q30201; 27 GO terms). Then we computed two percentages. The first one is the percentage of GO terms the hit has in common with HFE, the second one is the percentage of GO terms HFE has in common with the hit.<br />
* COPS classification: For each hit that could be mapped to a PDB ID we checked if it was within the same COPS cluster (L30-L99,S30,S90) as HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== BLAST ===<br />
<br />
<figtable id="blastdist"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_eval_blast_80.png|thumb|300px]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_ident_blast_80.png|thumb|300px]] <br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | <font size=1>'''Table 1:''' E-Value and identity distributions of the BLAST search against Big80.<br />
| <br />
|-<br />
|}<br />
</figtable><br />
<br />
The first BLAST search against the Big80 database reached the hit limit of 250 sequences with an e-Value of e-30 for the worst hit. So we did it again with a new limit of 1500 reported hits. These hits were then filtered for unique IDs and an e-Value cutoff of 2e-3. After the filtering 1159 hits were left.<br />
<br />
The distributions for the e-Values and identities of these hits are shown in <xr id="blastdist"/>. Most of the e-Values are between 1e-50 and 2e-3 (cutoff). Only few hits have a better e-Value. The identities are piled between 20% and 40% with two peaks at around 27% and 34% respectively.<br />
<br />
For the evaluation of the results we compared the hits' GO terms and COPS classifications. <xr id="blastgo"/> shows that the majority of the hits share almost all of their GO terms with the HFE protein. In contrast, only about 10% to 15% of HFE's GO terms are shared by most of the hits. This might be caused by the fact that most of the hit proteins didn't have as much GO terms as HFE (27 GO terms). The COPS classification (see <xr id="blastcops"/>) shows that most (59) of the 69 PDB entries share at least 80% structure similarity with HFE, but only a few have a high sequence similarity (which is in accordance with the previous identity statistic). Overall BLAST shows the best performance for finding proteins of similar structure (see <xr id="allcops"/>).<br />
<br />
<figtable id="blastgo"><br />
{| class="wikitable" style="float: left; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_blast-poogo.png|thumb|200px|Percentage of shared GO terms with HFE in relation to hit's GO terms.]]<br />
| align="right" | [[File:hemo_blast-pohfego.png|thumb|200px|Percentage of shared GO terms with HFE in relation to HFE's GO terms.]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="3" | <font size=1>'''Table 2:''' Common GO terms within BLAST hits.<br />
|-<br />
|}<br />
</figtable><br />
<br />
<figure id="blastcops"><br />
[[File:hemo_cops_blast.png|thumb|200px|left|<font size=1>'''Figure 1:''' COPS classification of BLAST hits. The figure shows the number of PDB IDs that are within the different similarity clusters with HFE.]]<br />
</figure><br />
<br />
<br style="clear:both;"><br />
<br />
=== PSI-BLAST ===<br />
<br />
<figtable id="psiblastdist"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_eval_psi_80.png|thumb|300px]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_ident_psi_80.png|thumb|300px]] <br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | <font size=1>'''Table 3:''' E-Value and identity distributions of the PSI-BLast search against Big80.<br />
| <br />
|-<br />
|}<br />
</figtable><br />
<br />
After the BLAST search, we also performed several searches with PSI-BLAST. This time we increased the number of reported hits and used a variety of parameter combinations to test their impact on the search results. The parameters to be changed were 'h' and 'j'. The first one, 'h', sets the e-Value cutoff for the inclusion of sequences into the PSI-BLAST-profile. The second one, 'j', is for the number of iterations for the PSI-BLAST search. For each of these parameters we used two different values: 2e-3 and 1e-10 for 'h', 2 and 10 for 'j'. This resulted in a total of 4 combinations.<br />
<br />
<br />
The first 4 searches were against Big80 with a maximum of 10000 reported hits. We also saved the PSI-BLAST profiles for later (see bellow). The hits for each individual parameter combination were again filtered for unique IDs and an e-Value cutoff of 2e-3. This resulted in the following number of hits:<br />
* h=2e-3, j=2: 1892 (2786 prefiltered)<br />
* h=2e-3, j=10: 1704 (2734 prefiltered)<br />
* h=1e-10, j=2: 2058 (3574 prefiltered)<br />
* h=1e-10, j=10: 2035 (3458 prefiltered)<br />
<xr id="psiblastdist"/> shows the e-Value and identity distributions for the Big80 results. In contrast to the previous BLAST search, we have more significant e-Values, but the identity shifts a bit to the left (lower identity). The differences between the parameter combinations are quite easy to spot. The lower e-Value cutoff (1-e10) also produces more significant hits (lower e-Values). This might be caused by the inclusion of fewer sequences into the profile and therefore a higher specificity for more closely related sequences (i.e. low e-Values). An increased number of iterations on the other hand reduces the number of significant hits and seems to slightly reduce the average identity.<br />
<br />
<br />
After the searches against Big80 we also ran PSI-BLAST against the Big database. We reused the profiles from the Big80 runs and also increased the maximum of reported hits to 100000.<br />
* h=2e-3, j=2: 23840 (25934 prefiltered)<br />
* h=2e-3, j=10: 25756 (30616 prefiltered)<br />
* h=1e-10, j=2: 26483 (28766 prefiltered)<br />
* h=1e-10, j=10: 27535 (29609 prefiltered)<br />
The two combinations with 10 iterations threw multiple error messages (but finished the process nevertheless). These errors were due to an internal code failure of PSI-BLAST and caused by too many possible hits[http://www.csc.fi/english/research/sciences/bioscience/programs/blast/optimized_psiblast].<br />
<br />
<br />
The performances of the different PSI-BLAST runs (see <xr id="psiblastruntime"/>) show that the cutoff for the profiles ('h') doesn't really affect the runtime. The number of iteration on the other hand has a big impact on the runtime. The size of the database, of course, also affects the runtime. The exceptionally high runtime for the 10-iteration runs against Big might also be caused by the errors mentioned above.<br />
<br />
<figtable id="psiblastruntime"><br />
{| class="wikitable", style="width:50%; border-collapse: collapse; border-style: solid; border-width:0px; border-color: #000"<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Iterations<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |2<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |2<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |10<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |10<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |E-Value<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |0.002<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |10E-10<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |0.002<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |10E-10<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Big80<br />
| style="border-style: solid; border-width: 0 0 0 0" |3m21<br />
| style="border-style: solid; border-width: 0 0 0 0" |3m6<br />
| style="border-style: solid; border-width: 0 0 0 0" |16m39<br />
| style="border-style: solid; border-width: 0 0 0 0" |16m41<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Big<br />
| style="border-style: solid; border-width: 0 0 0 0" |28m17<br />
| style="border-style: solid; border-width: 0 0 0 0" |26m43<br />
| style="border-style: solid; border-width: 0 0 0 0" |367m15<br />
| style="border-style: solid; border-width: 0 0 0 0" |64m4<br />
|-<br />
|}<br />
<font size=1>'''Table 4''': Runtime analysis of PSI-BLAST.<br />
</figtable><br />
<br />
<br />
The GO term and COPS classification analysis yields similar results to BLAST. The different parameter values have almost no effect on the GO terms. The only visible effect is that a higher iteration count seems to have a small negative effect on the GO term analysis. In the COPS analysis (see <xr id="psiblastcops"/>) the parameters seem to make almost no difference in the distribution of the different clusters (L30-L99,S30,S90), but a more strict e-Value cutoff and higher iteration count both have a negative effect on the overall percentages of hits within these clusters (see <xr id="allcops"/>).<br />
<br />
<figtable id="psiblastgo"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_psiblast-poogo.png|thumb|200px|Percentage of shared GO terms with HFE in relation to hit's GO terms (Big80).]]<br />
| align="right" | [[File:hemo_psiblast-pohfego.png|thumb|200px|Percentage of shared GO terms with HFE in relation to HFE's GO terms (Big80).]]<br />
|-<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_psiblastbig-poogo.png|thumb|200px|Percentage of shared GO terms with HFE in relation to hit's GO terms (Big).]]<br />
| align="right" | [[File:hemo_psiblastbig-pohfego.png|thumb|200px|Percentage of shared GO terms with HFE in relation to HFE's GO terms (Big).]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="2"| <font size=1>'''Table 5:''' Common GO terms within PSI-BLAST hits.<br />
|-<br />
|}<br />
</figtable><br />
<br />
<figtable id="psiblastcops"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_cops_psiblast.png|thumb|200px|Database: Big80]]<br />
|-<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_cops_psiblast_big.png|thumb|200px|Database: Big]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="2"| <font size=1>'''Table 6:''' COPS classification of PSI-BLAST hits.<br />
|-<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== HHblits ===<br />
<br />
<figtable id="hhblitsdist"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_eval_hhblits_80.png|thumb|300px]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_ident_hhblits_80.png|thumb|300px]] <br />
|-<br />
! scope="row" align="left" | <br />
| align="right" | <font size=1>'''Table 7:''' E-Value and identity distributions of the HHblits search against Uniprot20.<br />
| <br />
|-<br />
|}<br />
</figtable><br />
<br />
The final sequence search algorithm we apllied was HHblits. This time we searched against another database, Uniprot20. We set the number of reported hits (clusters) to 600 which corresponds to a worst e-Value of 0.0021. After filtering for unique clusters, we had 585 clusters left. Within these clusters we had 27588 unique Uniprot ACs. The most significant cluster with an e-Value of 2e-131 and an 40% identities accounted for 2771 (about 10% total) of these Uniprot ACs.<br />
<br />
In <xr id="hhblitsdist"/> you can see the distribution of the clusters' e-Values and identities. Like in the BLAST results, most e-Values are between 1e-50 and 2e-3. The majority of the identities are again between 20% and 40%, with a peak at around 26%.<br />
<br />
The runtime was 13m 13s. This means that HHblits' is more or less between BLAST's and PSI-BLAST's runtime.<br />
<br />
<br />
HHblits shows about the same results for the GO term analysis (<xr id="hhblitsgo"/>) regarding the percentage of HFE's GO terms. In contrast to the previous results, HHblits also has a high peak for the hit's GO terms at around 65% to 70%. This means that HHblits finds more proteins with a more distant related function than the other algorithms. The COPS classification (<xr id="hhblitscops"/> and <xr id="allcops"/>) is similar to PSI-BLAST (database: Big) in the L99 and L80 clusters, but HHblits has a better performance to find proteins in the L30, L40, and L60 clusters than PSI-BLAST (database: Big).<br />
<br />
<figtable id="hhblitsgo"><br />
{| class="wikitable" style="float: left; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_hhblits-poogo.png|thumb|200px|Percentage of shared GO terms with HFE in relation to hit's GO terms.]]<br />
| align="right" | [[File:hemo_hhblits-pohfego.png|thumb|200px|Percentage of shared GO terms with HFE in relation to HFE's GO terms.]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="2"| <font size=1>'''Table 8:''' Common GO terms within HHblits hits.<br />
|-<br />
|}<br />
</figtable><br />
<br />
<figure id="hhblitscops"><br />
[[File:hemo_cops_hhblits.png|thumb|200px|left|<font size=1>'''Figure 2:''' COPS classification of HHblits hits. The figure shows the number of PDB IDs that are within the different similarity clusters with HFE.]]<br />
</figure><br />
<br />
<br style="clear:both;"><br />
<br />
=== Comparison ===<br />
<br />
To compare the different search algorithms and PSI-BLAST combinations we collected the unique hits and created several Venn diagrams ([http://bioinfogp.cnb.csic.es/tools/venny/index.html Oliveros, J.C. (2007) VENNY. An interactive tool for comparing lists with Venn Diagrams.]). In order to compare PSI-BLAST and HHblits we took the runs of PSI-BLAST against the Big database. BLAST was never run against Big, so all of BLAST's hits are based on the Big80 database.<br />
<br />
<figtable id="psiblastoverlap"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_Overlap_PSI.png|thumb|200px|Database: Big80.]]<br />
| align="right" | [[File:hemo_Overlap_PSI_BIG.png|thumb|200px|Database: Big.]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="2"| <font size=1>'''Table 9:''' Overlap between the different PSI-BLAST runs.<br />
| <br />
|-<br />
|}<br />
</figtable><br />
<br />
First we compared the different variations of PSI-BLAST runs. <xr id="psiblastoverlap"/> shows that the different parameters have only a small effect on the results as most of them are shared between all combinations. Only the adjustment of the inclusion e-Value cutoff produces some hits unique to these configurations. This is true for both databases: Big80 and Big.<br />
<br />
<figtable id="alloverlap"><br />
{| class="wikitable" style="float: right; border: 2px solid darkgray;" cellpadding="2"<br />
! scope="row" align="left" | <br />
| align="right" | [[File:hemo_Overlap_All_BIG.png|thumb|200px|Databases: Big80, Big, Uniprot20.]]<br />
| align="right" | [[File:hemo_Overlap_PSI_HHB_BIG.png|thumb|200px|Databases: Big, Uniprot20.]]<br />
| align="right" | [[File:hemo_Overlap_PSI_BLAST.png|thumb|200px|Database: Big80.]]<br />
|-<br />
! scope="row" align="left" | <br />
| align="left" colspan="4"| <font size=1>'''Table 10:''' Overlap between the different search algorithms. BLAST results are always against the Big80 database, HHblits against Uniprot20, and <br />
PSI-BLAST against Big (left and middle figure) or Big80 (right figure).<br />
|<br />
|-<br />
|}<br />
</figtable><br />
<br />
When comparing all three algorithms (<xr id="alloverlap"/>), we can see that PSI-BLAST and BLAST have the most hits in common. This is not surprising as both algorithms use a similar approach to collect thier hits. HHblits and PSI-BLAST share about half of their hits. This seems also to be true for HHblits and BLAST, but the number of BLAST hits is too small to be sure.<br />
<br />
<figure id="allcops"><br />
[[File:hemo_copspercentages.png|thumb|200px|left|<font size=1>'''Figure 3:''' COPS classification statistics for all algorithms.]]<br />
</figure><br />
<br />
Last we compared the COPS analysis for all algorithms and PSI-BLAST combinations (see <xr id="allcops"/>). BLAST shows the best performance among all of them in the L99 and L80 cluster, though it is equal to PSI-BLAST (database: Big80) in the L30 to L60 clusters. When comparing the results for HHblits and PSI-BLAST (database: Big), HHblits yields slightly better results in the L30 to L60 range.<br />
<br />
<br style="clear:both;"><br />
<br />
== Multiple Sequence Alignments ==<br />
<br />
<br style="clear:both;"><br />
<br />
=== Dataset ===<br />
<br />
We created three groups of sequences for the MSAs. One group with only 40% (or lower) sequence identity to HFE, one with 60% identity or higher (max 99%), and a last one with 20% to 99% identity. We tried to get several sequences with PDB entries into the lists, but the only sequence with a identity higher than 40% was Q30201 (HFE) itself. In contrast, the whole 40% or lower group consists only of Uniprot ACs that can be mapped to PDB entries.<br />
Of course, every group also includes the HFE protein which means that there are really 3 groups with 11 sequences each.<br />
<br />
<br />
<figtable id="msagroup60"><br />
{| class="wikitable", style="width:70%; border-collapse: collapse; border-style: solid; border-width:0px; border-color: #000"<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Uniprot AC (Group 60-99)<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Identity<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Comment<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G3QU39<br />
| style="border-style: solid; border-width: 0 0 0 0" |99.14<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Gorilla gorilla gorilla<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |H2PI54<br />
| style="border-style: solid; border-width: 0 0 0 0" |97.41<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Pongo abelii<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q6B0J5<br />
| style="border-style: solid; border-width: 0 0 0 0" |95.98<br />
| style="border-style: solid; border-width: 0 0 0 0" |HFE protein OS=Homo sapiens<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G7P2L8<br />
| style="border-style: solid; border-width: 0 0 0 0" |94.54<br />
| style="border-style: solid; border-width: 0 0 0 0" |Putative uncharacterized protein OS=Macaca fascicularis<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F7GRH8<br />
| style="border-style: solid; border-width: 0 0 0 0" |90.52<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Callithrix jacchus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F7DKE9<br />
| style="border-style: solid; border-width: 0 0 0 0" |86.18<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Callithrix jacchus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q9GL42<br />
| style="border-style: solid; border-width: 0 0 0 0" |79.89<br />
| style="border-style: solid; border-width: 0 0 0 0" |Hereditary hemochromatosis protein homolog OS=Dicerorhinus sumatrensis<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F6RUG7<br />
| style="border-style: solid; border-width: 0 0 0 0" |78.16<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Equus caballus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G3THV5<br />
| style="border-style: solid; border-width: 0 0 0 0" |75.21<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein (Fragment) OS=Loxodonta africana<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G5BQE5<br />
| style="border-style: solid; border-width: 0 0 0 0" |67.66<br />
| style="border-style: solid; border-width: 0 0 0 0" |Hereditary hemochromatosis protein-like protein OS=Heterocephalus glaber<br />
|-<br />
|}<br />
</figtable><br />
<br />
<figtable id="msagroup40"><br />
{| class="wikitable", style="width:70%; border-collapse: collapse; border-style: solid; border-width:0px; border-color: #000"<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Uniprot AC (Group 00-40)<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Identity<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Comment<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P16391<br />
| style="border-style: solid; border-width: 0 0 0 0" |36.03<br />
| style="border-style: solid; border-width: 0 0 0 0" |RT1 class I histocompatibility antigen, AA alpha chain OS=Rattus norvegicus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P05534<br />
| style="border-style: solid; border-width: 0 0 0 0" |35.13<br />
| style="border-style: solid; border-width: 0 0 0 0" |HLA class I histocompatibility antigen, A-24 alpha chain OS=Homo sapiens<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q30597<br />
| style="border-style: solid; border-width: 0 0 0 0" |33.72<br />
| style="border-style: solid; border-width: 0 0 0 0" |MHC class I Mamu-A*02 (Fragment) OS=Macaca mulatta<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P01900<br />
| style="border-style: solid; border-width: 0 0 0 0" |32.11<br />
| style="border-style: solid; border-width: 0 0 0 0" |H-2 class I histocompatibility antigen, D-D alpha chain OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q31093<br />
| style="border-style: solid; border-width: 0 0 0 0" |31.74<br />
| style="border-style: solid; border-width: 0 0 0 0" |Histocompatibility 2, M region locus 3 OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P14432<br />
| style="border-style: solid; border-width: 0 0 0 0" |29.14<br />
| style="border-style: solid; border-width: 0 0 0 0" |H-2 class I histocompatibility antigen, TLA(B) alpha chain OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q860W6<br />
| style="border-style: solid; border-width: 0 0 0 0" |27.74<br />
| style="border-style: solid; border-width: 0 0 0 0" |Major histocompatibility complex class Ib M10.5 (Fragment) OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q31615<br />
| style="border-style: solid; border-width: 0 0 0 0" |26.38<br />
| style="border-style: solid; border-width: 0 0 0 0" |MHC class I H2-TL-27-129 mRNA (b haplotype), complete cds OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q31206<br />
| style="border-style: solid; border-width: 0 0 0 0" |25.87<br />
| style="border-style: solid; border-width: 0 0 0 0" |MHC class I H2-TL-T10-129 mRNA (b haplotype), complete cds OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P01921<br />
| style="border-style: solid; border-width: 0 0 0 0" |21.10<br />
| style="border-style: solid; border-width: 0 0 0 0" |H-2 class II histocompatibility antigen, A-D beta chain OS=Mus musculus<br />
|-<br />
|}<br />
</figtable><br />
<br />
<figtable id="msagroup00"><br />
{| class="wikitable", style="width:70%; border-collapse: collapse; border-style: solid; border-width:0px; border-color: #000"<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Uniprot AC (Group 00-99)<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Identity<br />
! style="text-align:left; border-style: solid; border-width: 0 0 2px 0" |Comment<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q6B0J5<br />
| style="border-style: solid; border-width: 0 0 0 0" |95.98<br />
| style="border-style: solid; border-width: 0 0 0 0" |HFE protein OS=Homo sapiens <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F7GRH8<br />
| style="border-style: solid; border-width: 0 0 0 0" |90.52<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Callithrix jacchus <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F7DKE9<br />
| style="border-style: solid; border-width: 0 0 0 0" |86.18<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Callithrix jacchus <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q9GL42<br />
| style="border-style: solid; border-width: 0 0 0 0" |79.89<br />
| style="border-style: solid; border-width: 0 0 0 0" |Hereditary hemochromatosis protein homolog OS=Dicerorhinus sumatrensis <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G5BQE5<br />
| style="border-style: solid; border-width: 0 0 0 0" |67.66<br />
| style="border-style: solid; border-width: 0 0 0 0" |Hereditary hemochromatosis protein-like protein OS=Heterocephalus glaber <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |G1PHG2<br />
| style="border-style: solid; border-width: 0 0 0 0" |57.43<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein (Fragment) OS=Myotis lucifugus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |F7C3B3<br />
| style="border-style: solid; border-width: 0 0 0 0" |40.11<br />
| style="border-style: solid; border-width: 0 0 0 0" |Uncharacterized protein OS=Macaca mulatta<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q30597<br />
| style="border-style: solid; border-width: 0 0 0 0" |33.72<br />
| style="border-style: solid; border-width: 0 0 0 0" |MHC class I Mamu-A*02 (Fragment) OS=Macaca mulatta <br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |Q860W6<br />
| style="border-style: solid; border-width: 0 0 0 0" |27.74<br />
| style="border-style: solid; border-width: 0 0 0 0" |Major histocompatibility complex class Ib M10.5 (Fragment) OS=Mus musculus<br />
|-<br />
| style="border-style: solid; border-width: 0 0 0 0" |P01921<br />
| style="border-style: solid; border-width: 0 0 0 0" |21.10<br />
| style="border-style: solid; border-width: 0 0 0 0" |H-2 class II histocompatibility antigen, A-D beta chain OS=Mus musculus<br />
|-<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== ClustalW ===<br />
==== ClustalW 0-40 ====<br />
<figure id="clustal040"><br />
[[File:hemo_msaClustalgroup0-40.png|View of the alignment of the 0-40 group aligned by ClustalW|thumb|600px|right]]<br />
</figure><br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 79<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01900<br />
| style="border-style: solid; border-width: 0"| 71<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 109<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31093<br />
| style="border-style: solid; border-width: 0"| 100<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31615<br />
| style="border-style: solid; border-width: 0"| 57<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P05534<br />
| style="border-style: solid; border-width: 0"| 71<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 171<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 88<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P16391<br />
| style="border-style: solid; border-width: 0"| 65<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P14432<br />
| style="border-style: solid; border-width: 0"| 52<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31206<br />
| style="border-style: solid; border-width: 0"| 30<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 23<br />
|}<br />
<!--<br />
79 Gaps gefunden! Bei Uniprot Q30597<br />
71 Gaps gefunden! Bei Uniprot P01900<br />
109 Gaps gefunden! Bei Uniprot Q860W6<br />
100 Gaps gefunden! Bei Uniprot Q31093<br />
57 Gaps gefunden! Bei Uniprot Q31615<br />
71 Gaps gefunden! Bei Uniprot P05534<br />
171 Gaps gefunden! Bei Uniprot P01921<br />
88 Gaps gefunden! Bei Uniprot Q30201<br />
65 Gaps gefunden! Bei Uniprot P16391<br />
52 Gaps gefunden! Bei Uniprot P14432<br />
30 Gaps gefunden! Bei Uniprot Q31206<br />
23 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
==== ClustalW 0-99 ====<br />
<figure id="clustal099"><br />
[[File:hemo_msaclustalgroup0-99.png||View of the alignment of the 0-99 group aligned by ClustalW|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 42<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 72<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 32<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 51<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G1PHG2<br />
| style="border-style: solid; border-width: 0"| 61<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 54<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 134<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 51<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7C3B3<br />
| style="border-style: solid; border-width: 0"| 146<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 62<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 51<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 21<br />
|}<br />
<br />
<!--<br />
42 Gaps gefunden! Bei Uniprot Q30597<br />
72 Gaps gefunden! Bei Uniprot Q860W6<br />
32 Gaps gefunden! Bei Uniprot G5BQE5<br />
51 Gaps gefunden! Bei Uniprot Q9GL42<br />
61 Gaps gefunden! Bei Uniprot G1PHG2<br />
54 Gaps gefunden! Bei Uniprot Q6B0J5<br />
134 Gaps gefunden! Bei Uniprot P01921<br />
51 Gaps gefunden! Bei Uniprot Q30201<br />
146 Gaps gefunden! Bei Uniprot F7C3B3<br />
62 Gaps gefunden! Bei Uniprot F7DKE9<br />
51 Gaps gefunden! Bei Uniprot F7GRH8<br />
21 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
==== ClustalW 60-99 ====<br />
<figure id="clustal6099"><br />
[[File:hemo_msaclustalgroup60-99.png||View of the alignment of the 60-99 group aligned by ClustalW|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3THV5<br />
| style="border-style: solid; border-width: 0"| 12<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| H2PI54<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F6RUG7<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 6<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 28<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3QU39<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 36<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G7P2L8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 175<br />
|}<br />
<br />
<br />
<!--<br />
12 Gaps gefunden! Bei Uniprot G3THV5<br />
25 Gaps gefunden! Bei Uniprot H2PI54<br />
25 Gaps gefunden! Bei Uniprot F6RUG7<br />
6 Gaps gefunden! Bei Uniprot G5BQE5<br />
25 Gaps gefunden! Bei Uniprot Q9GL42<br />
28 Gaps gefunden! Bei Uniprot Q6B0J5<br />
25 Gaps gefunden! Bei Uniprot Q30201<br />
25 Gaps gefunden! Bei Uniprot G3QU39<br />
25 Gaps gefunden! Bei Uniprot F7GRH8<br />
36 Gaps gefunden! Bei Uniprot F7DKE9<br />
25 Gaps gefunden! Bei Uniprot G7P2L8<br />
175 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
<br />
<br style="clear:both;"><br />
<br />
=== Muscle ===<br />
<br />
==== Muscle Group 0-40 ====<br />
<figure id="muscle040"><br />
[[File:hemo_msamusclegroup0-40.png|View of the alignment of the 0-40 group aligned by Muscle|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01900<br />
| style="border-style: solid; border-width: 0"| 82<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 90<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31093<br />
| style="border-style: solid; border-width: 0"| 111<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 120<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31615<br />
| style="border-style: solid; border-width: 0"| 68<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P05534<br />
| style="border-style: solid; border-width: 0"| 82<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 99<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 182<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P16391<br />
| style="border-style: solid; border-width: 0"| 76<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P14432<br />
| style="border-style: solid; border-width: 0"| 63<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31206<br />
| style="border-style: solid; border-width: 0"| 41<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 22<br />
|}<br />
<!--<br />
<br />
<br />
Muscle Group 0-40<br />
82 Gaps gefunden! Bei Uniprot P01900<br />
90 Gaps gefunden! Bei Uniprot Q30597<br />
111 Gaps gefunden! Bei Uniprot Q31093<br />
120 Gaps gefunden! Bei Uniprot Q860W6<br />
68 Gaps gefunden! Bei Uniprot Q31615<br />
82 Gaps gefunden! Bei Uniprot P05534<br />
99 Gaps gefunden! Bei Uniprot Q30201<br />
182 Gaps gefunden! Bei Uniprot P01921<br />
76 Gaps gefunden! Bei Uniprot P16391<br />
63 Gaps gefunden! Bei Uniprot P14432<br />
41 Gaps gefunden! Bei Uniprot Q31206<br />
22 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
==== Muscle Group 0-99 ====<br />
<figure id="muscle099"><br />
[[File:hemo_msamusclegroup0-99.png|View of the alignment of the 0-99 group aligned by Muscle|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 44<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 74<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 34<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 53<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 56<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G1PHG2<br />
| style="border-style: solid; border-width: 0"| 63<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 53<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 136<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7C3B3<br />
| style="border-style: solid; border-width: 0"| 148<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 64<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 53<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 22<br />
|}<br />
<br />
<!--<br />
Muscle Group 0-99<br />
44 Gaps gefunden! Bei Uniprot Q30597<br />
74 Gaps gefunden! Bei Uniprot Q860W6<br />
34 Gaps gefunden! Bei Uniprot G5BQE5<br />
53 Gaps gefunden! Bei Uniprot Q9GL42<br />
56 Gaps gefunden! Bei Uniprot Q6B0J5<br />
63 Gaps gefunden! Bei Uniprot G1PHG2<br />
53 Gaps gefunden! Bei Uniprot Q30201<br />
136 Gaps gefunden! Bei Uniprot P01921<br />
148 Gaps gefunden! Bei Uniprot F7C3B3<br />
64 Gaps gefunden! Bei Uniprot F7DKE9<br />
53 Gaps gefunden! Bei Uniprot F7GRH8<br />
22 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
==== Muscle Group 60-99 ====<br />
<figure id="muscle06099"><br />
[[File:hemo_msamusclegroup60-99.png|View of the alignment of the 60-99 group aligned by Muscle|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3THV5<br />
| style="border-style: solid; border-width: 0"| 12<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| H2PI54<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F6RUG7<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 6<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 28<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3QU39<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 36<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G7P2L8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 175<br />
|}<br />
<br />
<br />
<!--Muscle Group 60-99<br />
12 Gaps gefunden! Bei Uniprot G3THV5<br />
25 Gaps gefunden! Bei Uniprot H2PI54<br />
25 Gaps gefunden! Bei Uniprot F6RUG7<br />
6 Gaps gefunden! Bei Uniprot G5BQE5<br />
25 Gaps gefunden! Bei Uniprot Q9GL42<br />
28 Gaps gefunden! Bei Uniprot Q6B0J5<br />
25 Gaps gefunden! Bei Uniprot Q30201<br />
25 Gaps gefunden! Bei Uniprot G3QU39<br />
25 Gaps gefunden! Bei Uniprot F7GRH8<br />
36 Gaps gefunden! Bei Uniprot F7DKE9<br />
25 Gaps gefunden! Bei Uniprot G7P2L8<br />
175 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
<br />
<br style="clear:both;"><br />
<br />
=== T-Coffee ===<br />
==== T-Coffee Group 0-40 ====<br />
<figure id="tcoffee040"><br />
[[File:hemo_msatcoffeegroup0-40.png|View of the alignment of the 0-40 group aligned by T-Coffee|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 92<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01900<br />
| style="border-style: solid; border-width: 0"| 84<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31093<br />
| style="border-style: solid; border-width: 0"| 113<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 122<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31615<br />
| style="border-style: solid; border-width: 0"| 70<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P05534<br />
| style="border-style: solid; border-width: 0"| 84<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 184<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 101<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P14432<br />
| style="border-style: solid; border-width: 0"| 65<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P16391<br />
| style="border-style: solid; border-width: 0"| 78<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31206<br />
| style="border-style: solid; border-width: 0"| 43<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 22<br />
|}<br />
<!--<br />
<br />
T-Coffee Group 0-40<br />
92 Gaps gefunden! Bei Uniprot Q30597<br />
84 Gaps gefunden! Bei Uniprot P01900<br />
113 Gaps gefunden! Bei Uniprot Q31093<br />
122 Gaps gefunden! Bei Uniprot Q860W6<br />
70 Gaps gefunden! Bei Uniprot Q31615<br />
84 Gaps gefunden! Bei Uniprot P05534<br />
184 Gaps gefunden! Bei Uniprot P01921<br />
101 Gaps gefunden! Bei Uniprot Q30201<br />
65 Gaps gefunden! Bei Uniprot P14432<br />
78 Gaps gefunden! Bei Uniprot P16391<br />
43 Gaps gefunden! Bei Uniprot Q31206<br />
22 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
====T-Coffee Group 0-99 ====<br />
<figure id="tcoffee099"><br />
[[File:hemo_msatcoffeegroup0-99.png|View of the alignment of the 0-99 group aligned by T-Coffee|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 51<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 81<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 41<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 60<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 63<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G1PHG2<br />
| style="border-style: solid; border-width: 0"| 70<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 143<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 60<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7C3B3<br />
| style="border-style: solid; border-width: 0"| 155<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 71<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 60<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 22<br />
|}<br />
<br />
<!--<br />
T-Coffee Group 0-99<br />
51 Gaps gefunden! Bei Uniprot Q30597<br />
81 Gaps gefunden! Bei Uniprot Q860W6<br />
41 Gaps gefunden! Bei Uniprot G5BQE5<br />
60 Gaps gefunden! Bei Uniprot Q9GL42<br />
63 Gaps gefunden! Bei Uniprot Q6B0J5<br />
70 Gaps gefunden! Bei Uniprot G1PHG2<br />
143 Gaps gefunden! Bei Uniprot P01921<br />
60 Gaps gefunden! Bei Uniprot Q30201<br />
155 Gaps gefunden! Bei Uniprot F7C3B3<br />
71 Gaps gefunden! Bei Uniprot F7DKE9<br />
60 Gaps gefunden! Bei Uniprot F7GRH8<br />
22 komplett übereinstimmende Positionen (Conserved)<br />
<br />
--><br />
==== T-Coffee Group 60-99 ====<br />
<figure id="tcoffee6099"><br />
[[File:hemo_msatcoffeegroup60-99.png|View of the alignment of the 60-99 group aligned by T-Coffee|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3THV5<br />
| style="border-style: solid; border-width: 0"| 12<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| H2PI54<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F6RUG7<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G5BQE5<br />
| style="border-style: solid; border-width: 0"| 6<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q9GL42<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q6B0J5<br />
| style="border-style: solid; border-width: 0"| 28<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G3QU39<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| G7P2L8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7GRH8<br />
| style="border-style: solid; border-width: 0"| 25<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| F7DKE9<br />
| style="border-style: solid; border-width: 0"| 25<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 175<br />
|}<br />
<br />
<br />
<!--<br />
T-Coffee Group 60-99<br />
12 Gaps gefunden! Bei Uniprot G3THV5<br />
25 Gaps gefunden! Bei Uniprot H2PI54<br />
25 Gaps gefunden! Bei Uniprot F6RUG7<br />
6 Gaps gefunden! Bei Uniprot G5BQE5<br />
25 Gaps gefunden! Bei Uniprot Q9GL42<br />
28 Gaps gefunden! Bei Uniprot Q6B0J5<br />
25 Gaps gefunden! Bei Uniprot Q30201<br />
25 Gaps gefunden! Bei Uniprot G3QU39<br />
25 Gaps gefunden! Bei Uniprot G7P2L8<br />
25 Gaps gefunden! Bei Uniprot F7GRH8<br />
36 Gaps gefunden! Bei Uniprot F7DKE9<br />
175 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
<br />
<br style="clear:both;"><br />
<br />
==== 3D-Coffee ====<br />
==== T-Coffee Group 0-40 ====<br />
<figure id="3dcoffee040"><br />
[[File: hemo_msa3dcoffeegroup0-40.png|View of the alignment of the 0-40 group aligned by 3D-Coffee|thumb|600px|right]]<br />
</figure><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Sequence<br />
! style="border-style: solid; border-width: 0 0 1px 0"| Gap<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30597<br />
| style="border-style: solid; border-width: 0"| 92<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01900<br />
| style="border-style: solid; border-width: 0"| 84<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31093<br />
| style="border-style: solid; border-width: 0"| 113<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q860W6<br />
| style="border-style: solid; border-width: 0"| 122<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31615<br />
| style="border-style: solid; border-width: 0"| 70<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P05534<br />
| style="border-style: solid; border-width: 0"| 84<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P01921<br />
| style="border-style: solid; border-width: 0"| 184<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q30201<br />
| style="border-style: solid; border-width: 0"| 101<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P14432<br />
| style="border-style: solid; border-width: 0"| 65<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| P16391<br />
| style="border-style: solid; border-width: 0"| 78<br />
|-<br />
| style="border-style: solid; border-width: 0 1px 0 0"| Q31206<br />
| style="border-style: solid; border-width: 0"| 43<br />
|}<br />
<br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"<br />
|-<br />
! style="border-style: solid; border-width: 0 1px 1px 0"| Conserved<br />
|-<br />
! style="border-style: solid; border-width: 0 0 1px 0"| 22<br />
|}<br />
<!--<br />
<br />
3DCoffee Group 0-40<br />
92 Gaps gefunden! Bei Uniprot Q30597<br />
84 Gaps gefunden! Bei Uniprot P01900<br />
113 Gaps gefunden! Bei Uniprot Q31093<br />
122 Gaps gefunden! Bei Uniprot Q860W6<br />
70 Gaps gefunden! Bei Uniprot Q31615<br />
84 Gaps gefunden! Bei Uniprot P05534<br />
184 Gaps gefunden! Bei Uniprot P01921<br />
101 Gaps gefunden! Bei Uniprot Q30201<br />
65 Gaps gefunden! Bei Uniprot P14432<br />
78 Gaps gefunden! Bei Uniprot P16391<br />
43 Gaps gefunden! Bei Uniprot Q31206<br />
22 komplett übereinstimmende Positionen (Conserved)<br />
--><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Conservation of important positions ===<br />
<br />
{| style=" border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0;class=wikitable"<br />
|-<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | Position<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | 3DCoffee0-40<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | T-Coffee0-40<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | T-Coffee0-99<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | T-Coffee60-99<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | Muscle0-40<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | Muscle0-99<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | Muscle60-99<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | ClustalW0-40<br />
! style="text-align:left; border-style: solid; border-width: 0px 1px 1px 0px;scope=col" | ClustalW0-99<br />
! style="text-align:left; border-style: solid; border-width: 0px 0px 1px 0px;scope=col" |ClustalW60-99<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col" scope="row"| 110<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 9/10(Gap)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 9/10(Gap)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 8/10(Gap)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 10/10<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 9/10(Gap)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 8/10(Gap)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 10/10<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 9/10(Mismatch)<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 8/10(Gap/Mismatch)<br />
|style="text-align:center; border-style: solid; border-width: 0px 0px 0px 0;scope=col"|10/10<br />
|-<br />
!style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 130<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 10/10<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 10/10<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| X<br />
| style="text-align:center; border-style: solid; border-width: 0px 0px 0px 0;scope=col"|10/10<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 234<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|X<br />
| style="text-align:center; border-style: solid; border-width: 0px 0px 0px 0;scope=col"|10/10<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"| 124<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|9/10(Gap)<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|9/10(Gap)<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|9/10(Gap)<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 0px 0px 0;scope=col"|10/10<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 187<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
|style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"| 10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 0px 0px 0;scope=col"|10/10<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"| 225<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|9/10(Mismatch)<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 1px 0px 0px 0;scope=col"|10/10<br />
|-<br />
! style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|282<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 1px 0px 0;scope=col"|10/10<br />
| style="text-align:center; border-style: solid; border-width: 0px 0px 0px 0;scope=col"|10/10<br />
|}<br />
<br />
All positions are some at which the protein is modified.<br />
<br />
<br />
The positions are not ordered by their appearance because of their type of modification.<br />
The first three noted positions (110, 130, 234) are positions which are modified via glycosylation. The last four are responsible for disulfide-bonds (as they're grouped together, position 124 builds a disulfide bond with position 187, the same goes for positions 225 and 282).<br />
<br />
A X represents a non-conserved position within the alignment, meaning that 5 or less sequences have the same amino acid as the HFE sequence at that position.<br />
<br />
One can see that in nearly all groups and in all alignments the disulfide positions are conserved. This may show that the disulfide bond is crucial for the 3d structure of the protein and therefore its function.<br />
Of the Glycosylation positions only position 110 seems to be quite conserved, meaning it has a higher probability to be important than the other glycolysation positions.<br />
<br />
The conservation of all these positions when looking at the 60-99 groups can be explained by the high similarity of the proteins.<br />
<br />
Also one should keep in mind that these statistics are withdrawn from 10 proteins, and due to random selection of these errors may have occured.<br />
<br />
When looking at the domain region(positions 207-298) one can see in the MSAs that this region in all groups seems to be highly conserved. Even in the group with 20-40% identity. This is what we expected because the methods should find similar proteins and as we know mutations that last occur less often in functional domains.</div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28578Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:57:02Z<p>Joerdensv: /* Protein-Water */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
Continuation of [[Molecular_Dynamics_Simulations_Hemochromatosis|task 8]]. Analysis of the MD simulations for the wildtype and two mutants (R224W and C282S).<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells| Minimum distance between periodic boundary cells]].<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (see <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the values of the following timesteps) lower at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmful mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28577Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:56:01Z<p>Joerdensv: /* Protein-Water */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
Continuation of [[Molecular_Dynamics_Simulations_Hemochromatosis|task 8]]. Analysis of the MD simulations for the wildtype and two mutants (R224W and C282S).<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells| Minimum distance between periodic boundary cells]].<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
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T217I<br />
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Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
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Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
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Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
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gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
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R224W<br />
Checking file scwrl_R224W_md.xtc<br />
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# Atoms 68602<br />
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gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (see <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the values of the following timesteps) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmful mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28572Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:50:59Z<p>Joerdensv: /* Molecular dynamics simulations for HFE */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
Continuation of [[Molecular_Dynamics_Simulations_Hemochromatosis|task 8]]. Analysis of the MD simulations for the wildtype and two mutants (R224W and C282S).<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells| Minimum distance between periodic boundary cells]].<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
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Step 2001 5<br />
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Velocities 0<br />
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Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
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# Atoms 68607<br />
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Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
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Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
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<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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Velocities 0<br />
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Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (see <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmful mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Hemochromatosis_2012&diff=28569Hemochromatosis 20122012-08-31T19:39:51Z<p>Joerdensv: /* HFE Gene */</p>
<hr />
<div>== Summary ==<br />
Hereditary hemochromatosis is an autosomal recessive monogenetic metabolic disease caused by mutations in the HFE gene. Malfunction of the HFE gene leads to an increased absorption of iron in the intestines. Hereditary hemochromatosis was first described in a diabetes report by Armand Trousseau in 1865.<br />
<br />
The high iron concentration within the body can cause several side-effects such as arthritis, diabetes, heart muscle disorders, hepatic cirrhosis, hyperpigmentation, and hypopituitarism. The accumulation of iron is a slow process and therefore most patients begin to show symptoms between the age of 30 and 60.<br />
<br />
Although this site focuses on hereditary hemochromatosis (hemochromatosis type 1) it is worth noting, that there are differrent types of hemochromatosis.<br />
* Hereditary hemochromatosis (type 1) - autosomal recessive; gene: HFE<br />
* Juvenile hemochromatosis (type 2A and type 2B) - autosomal recessive; genes: HJV (2A) / HAMP (2B)<br />
* Hemochromatosis type 3 - autosomal recessive; gene: TFR2<br />
* Hemochromatosis type 4 - autosomal dominant; gene: SLC40A1<br />
<br />
<br />
=== Cross-references ===<br />
* [http://www.omim.org/entry/235200 Hemochromatosis type 1 (OMIM)]<br />
* [http://www.omim.org/entry/602390 Hemochromatosis type 2A (OMIM)]<br />
* [http://www.omim.org/entry/613313 Hemochromatosis type 2B (OMIM)]<br />
* [http://www.omim.org/entry/604250 Hemochromatosis type 3 (OMIM)]<br />
* [http://www.omim.org/entry/606069 Hemochromatosis type 4 (OMIM)]<br />
<br />
== Prevalence ==<br />
Hereditary hemochromatosis is quite common, especially in groups with european ancestry. It has a prevalence of about 2 to 3 in 1000 and a carrier rate of about 10 percent.<br />
<br />
== Phenotype ==<br />
Hereditary hemochromatosis leads to an unregulated absorption of dietary iron. Due to the increased concentration of iron within the body other dysfunctions can occur depending on the tissue.<br />
<br />
* The accumulation of iron causes a brown coloring of the skin (hyperpigmentation). This can be seen especially well in the armpits, but also on internal organs such as the liver.<br />
* Iron deposits in the joints may lead to arthritis.<br />
* A high concentration of iron in the pituitary gland decreases the secretion of hormones (hypopituitarism). This may cause further effects like testicle dysfunction and the loss of sex drive.<br />
* Regarding the liver it may lead to liver cirrhosis, within the heart to heart muscle disorders (cardiomyopathy).<br />
* Iron also aids the creation of free radicals which may damage the DNA and cause cancer.<br />
* In the blood iron competes with chromium for the binding to transferrin. Chromium is important for the normal function of insulin. This disturbance of chromium (due to too much iron) may lead to diabetes.<br />
<br />
Men show these symptoms more often than women. This is due to the loss of blood (and iron) during menstruation and pregnancy. The rate of iron accumulation also depends on the patient's diet. Most patients begin to show symptoms between the age of 30 and 60.<br />
<br />
=== Cross-references ===<br />
See also description of this disease in: <br />
* [http://en.wikipedia.org/wiki/Haemochromatosis_type_1 Wikipedia (english)]<br />
* [http://de.wikipedia.org/wiki/Hämochromatose Wikipedia (german)]<br />
* [http://www.omim.org/entry/235200 OMIM]<br />
<br />
==HFE Gene==<br />
<br />
<br />
<figure id="fig1"><br />
[[File:HFE-gene.png|frame|<font size=1>'''Figure 1:''' Homo sapiens chromosome 6 with marked HFE gene locus (source: genecards)]]<br />
</figure><br />
<br />
The HFE gene is located on the short arm of the sixth chromosome (cf. <xr id="fig1"/>).<br />
It has a length of 11,124 bases and lies on the plus strand. It further contains six exons, coding for the protein, with a length of 1047 bases.<br />
<br />
===HFE protein===<br />
The protein coded by the HFE gene consists of 4 chains, with 2 different entities in total, the HFE chain and the Beta-2-Microglobulin chain. The conformation of HFE itself can be seen in <xr id="fig2"/>, in complex with Transferrin in <xr id="fig3"/>.<br />
====HFE chain====<br />
The HFE chain of the protein consists of 8 helices, encoded by 70 residues, and 20 strands, coded by 109 residues.<br />
====Beta-2-Microglobulin====<br />
The Beta-2-Microglobulin chain consists of 11 strands, which are encoded by 51 residues.<br />
<br />
<figure id="fig2"><br />
[[File:HFE.jpg|thumb|100px|left|<font size=1>'''Figure 2:''' HFE protein (source: [http://www.rcsb.org/pdb/explore.do?structureId=1A6Z PDB])]]<br />
</figure><br />
<br />
<figure id="fig3"><br />
[[File:HFETransferrin.jpg|thumb|center|100px|<font size=1>'''Figure 3:''' HFE-Transferrin complex (source: [http://www.rcsb.org/pdb/explore.do?structureId=1DE4 PDB])]]<br />
</figure><br />
<br />
<br />
===Cross-references===<br />
* [http://www.genecards.org/cgi-bin/carddisp.pl?gene=HFE genecards]<br />
* [http://www.genome.jp/dbget-bin/www_bget?hsa:3077 KEGG]<br />
<br />
== Biochemical disease mechanism ==<br />
<br />
<figure id="fig4"><br />
[[File:genetic1.gif|thumb|150px|right|<font size=1>'''Figure 4:''' Iron absorption pathway ([http://home.vicnet.net.au/~johnlee/Hemo/genetic.htm source]). See figure for a detailed description.]]<br />
</figure><br />
<br />
Under normal circumstances, iron is absorbed in the intestines and bound to transferrin (cf. <xr id="fig4"/>). This iron loaded transferrin then associates with the transferrin receptor (TFRC) for further transport through the body. HFE forms a complex with TFRC to reduce its affinity for iron-loaded transferrin and to partially inhibit the release of the iron once the complex reaches the target cells. Further it seems that HFE plays a role in a sensory pathway to determine the iron concentration within the body and to regulate the absorption of iron through the expression of DMT1 (divalent metal transporter).<br />
<br />
<figure id="fig5"><br />
[[File:genetic2.gif|thumb|150px|right|<font size=1>'''Figure 5:''' HFE mutation effects ([http://home.vicnet.net.au/~johnlee/Hemo/genetic.htm source]). See figure for a detailed description.]]<br />
</figure><br />
<br />
The different mutations in the HFE gene diminish or inhibit these functions (cf. <xr id="fig5"/>). C282Y affects the beta-2-microglobulin binding site and prevents the formation of the HFE-TFRC-complex. H63D does not prevent the formation of the complex, however reduces the inhibitory functions of HFE. This causes the increased iron uptake and accumulation of iron within the cells which is associated with hereditary hemochromatosis.<br />
<br />
Dysfunctional HFE seems to also lead to a lack of hepcidin, another protein that (down-)regulates the iron uptake.<br />
<br />
<br />
=== Cross-references ===<br />
* [http://home.vicnet.net.au/~johnlee/Hemo/genetic.htm Protein involved in iron absorption]<br />
<br />
<br style="clear:both;"><br />
<br />
== Mutations ==<br />
===Inheritance===<br />
As hereditary hemochromatosis is an autosomal recessive disease, the genes on both chromosomes have to be affected by a mutation/mutations to lead to an outbreak of the disease. This can be through homozygosity or compound heterozygosity. However, in 30-50% of the cases the patients didn't show any symptoms of an iron overload.<br />
<br />
The most common genotypes for hemochromatosis are:<br />
* C282Y homozygosity<br />
* C282Y/H63D compound heterozygosity<br />
* H63D homozygosity.<br />
<br />
===Reference sequence===<br />
KEGG lists the following sequence for the HFE protein:<br />
<br />
MGPRA<font color="red">R</font>PALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGY<font color="red">V</font>DDQLF<font color="red">V</font>F<br />
YD<font color="red">H</font>E<font color="red">S</font>RRVEP<font color="red">R</font>TPWVSSRISSQMWLQLSQSLK<font color="red">G</font>WDHMFTVDFWT<font color="red">I</font>MENHNHSKESHTLQV<br />
ILGCEM<font color="red">Q</font>EDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKL<font color="red">EW</font>ERHKIR<font color="red">A</font>RQNR<br />
AY<font color="red">L</font>ERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTL<font color="red">R</font>CRALNYYPQNITMKWL<br />
KDKQPMDAKEFEPKDVLPNGDGTYQGWITLA<font color="red">V</font>PPGEEQRYT<font color="red">CQ</font>VEHPGLDQPLI<font color="red">V</font>IWEPS<br />
PSGTLVIGVISGIAVFVVILFIGILFIIL<font color="red">R</font>KRQGSRGAMGHYVLAERE<br />
This protein has a length of 348 amino acids and the red positions indicate the positions of known mutations, associated with hemochromatosis.<br />
<br />
=== Disease causing mutations ===<br />
<br />
Arg-Ser 6<br />
Val Met 53<br />
Val-Met 59<br />
His-His 63<br />
His-Asp 63<br />
Ser-Cys 65<br />
<!-- Arg-Cys 66 --><br />
Arg-Term 71<br />
Gly-Arg 93<br />
Ile-Thr 105<br />
Gln-His 127<br />
Glu-Gln 168<br />
Glu-Term 168<br />
Trp-Term 169<br />
Ala-Val 176<br />
Leu-Pro 183<br />
Arg-Gln 224<br />
Val-Leu 272<br />
<!-- Glu-Lys 277 --><br />
Cys-Tyr 282<br />
Cys-Ser 282<br />
Gln-Pro 283<br />
Val-Ala 295<br />
Arg-Met 330<br />
the mutations are listed as fromAA-toAA atPosition#<br />
<br />
====Deletions====<br />
=====small=====<br />
AGTCGC^67CGTGtGGAGCCCCGA<br />
AGTCTG^92AAAGgGTGGGATCAC<br />
ATTGG^156AGAGCaGCAGAACCCA<br />
GCAGCA^159GAACcCAGGGCCTGG<br />
=====gross=====<br />
22 bp cd 50 nt.370 (described at genomic DNA level)<br />
32744 bp incl. entire gene [Alu mediated] (described at genomic DNA level)<br />
<br />
====Insertions====<br />
GGACC^264TACCAaGGGCTGGATA<br />
<br />
===Disease causing splicings===<br />
IVS2 ds +4 T-C<br />
IVS3 ds +1 G-T<br />
IVS5 ds +1 G-A<br />
<br />
===Complex rearrangements===<br />
c.[128G>A;187C>G], p.[G43D;H63D]<br />
<br />
===Cross-references===<br />
[http://www.hgmd.cf.ac.uk/ac/gene.php?gene=HFE HGMD]<br />
<br />
== Diagnosis ==<br />
Diagnosis of hereditary hemochromatosis can be achieved by multiple methods.<br />
<br />
* '''Blood test:''' Measurement of the transferrin saturation or the serum ferritin concentration in the blood. Values that are significantly above the normal levels can be an indicator for hemochromatosis.<br />
* '''Liver biopsy:''' Extraction and examination of liver tissue. A high concentration of iron within the liver corresponds to a high concentration of iron in the body.<br />
* '''MRI:''' Magnetic resonance imaging can also be used to determine the iron concentration within the liver.<br />
<br />
If these methods show a risk for hemochromatosis, a genetic analysis for the known HFE mutations is recommended.<br />
<br />
=== Cross-references ===<br />
* [http://www.medicinenet.com/iron_overload/page3.htm Iron Overload: Diagnosis]<br />
<br />
== Treatment ==<br />
Hemochromatosis itself cannot be treated, but the symptoms can be prevented or lessened.<br />
<br />
* '''Phlebotomy:''' Bloodletting can be used to decrease the iron concentration. About 500ml of blood are extracted once a week until the iron concentration reaches a normal level. The extractions should then be repeated every two to three months.<br />
* '''Drugs:''' If the patient can’t handle frequent blood loss, chelating agents can be used in order to bind the iron in the blood and increase its excretion through feces and urine. Examples are deferoxamine, deferasirox, and deferiprone.<br />
* '''Diet:''' Reducing the consumption of iron rich food decreases the rate of iron accumulation and therefore the development of the symptoms. It is also helpful to avoid the consumption of vitamin C about two hours before or after meals, because vitamin C assists the absorption of iron. Alcohol should also be avoided.<br />
<br />
All of these treatments have to be continued throughout life to keep the iron concentration within normal levels.<br />
<br />
=== Cross-references ===<br />
* [http://www.medicinenet.com/iron_overload/page4.htm Iron Overload: Treatment]<br />
* [http://www.medscape.com/viewarticle/405860_8 Hereditary Hemochromatosis: Treatment]<br />
<br />
== References ==<br />
External links:<br />
* [http://en.wikipedia.org/wiki/Iron_overload Iron overload (en.wikipedia.org)]<br />
* [http://en.wikipedia.org/wiki/Haemochromatosis_type_1 Hemochromatosis type 1 (en.wikipedia.org)]<br />
* [http://de.wikipedia.org/wiki/Hämochromatose Hämochromatose (de.wikipedia.org)]<br />
* [http://www.omim.org/entry/235200 OMIM entry 235200 (Hemochromatosis)]<br />
* [http://www.omim.org/entry/613609 OMIM entry 613609 (HFE gene)]<br />
* [http://www.uniprot.org/uniprot/Q30201 Uniprot Q30201 (HFE gene)]<br />
* [http://www.americanhs.org/ American Hemochromatosis Society]<br />
<br />
== Tasks ==<br />
* Task 2: [[Sequence Alignments Hemochromatosis|Sequence alignments]]<br />
* Task 3: [[Sequence-Based Predictions Hemochromatosis|Sequence-based predictions]]<br />
* Task 4: [[Homology Based Structure Predictions Hemochromatosis|Homology based structure predictions]]<br />
* Task 5: [[Researching And Mapping Point Mutations Hemochromatosis|Researching and mapping point mutations]]<br />
* Task 6: [[Sequence-Based Mutation Analysis Hemochromatosis|Sequence-based mutation analysis]]<br />
* Task 7: [[Structure-Based Mutation Analysis Hemochromatosis|Structure-based mutation analysis]]<br />
* Task 8: [[Molecular Dynamics Simulations Hemochromatosis|Molecular dynamics simulations]]<br />
* Task 9: [[Normal Mode Analysis Hemochromatosis|Normal mode analysis]]<br />
* Task 10: [[Molecular Dynamics Simulations Analysis Hemochromatosis|Molecular dynamics simulations analysis]]</div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28559Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:21:11Z<p>Joerdensv: /* Protein-Water */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (see <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmfull mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28558Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:20:48Z<p>Joerdensv: /* hydrogen-bonds between protein and protein / protein and water */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases (see <xr id="tab:hbonds_pp"/>) the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (compare section [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Protein-Protein|Protein-Protein]]), the bonds formed with hydrogen show a different behavior (compare <xr id="tab:hbonds_pw"/>).<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmfull mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28555Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:15:16Z<p>Joerdensv: /* solvent accessible surface area */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Velocities 0<br />
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Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
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Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
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Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmfull mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28554Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:14:55Z<p>Joerdensv: /* solvent accessible surface area */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs (see <xr id="tab:sas"/>) show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen (<xr id="tab:sas_res"/>).<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmfull mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations (5+) should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28552Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:13:31Z<p>Joerdensv: /* Radius of gyration */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
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T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
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Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Box 2001 5<br />
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gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
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C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
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Box 2001 5<br />
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gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
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R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
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<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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# Atoms 68603<br />
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Box 2001 5<br />
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gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
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Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs (<xr id="tab:gyration_ca"/> and <xr id="tab:gyration_prot"/>) show the radii of gyration in general as well as in each dimension. From start to end there is a slight increase in general gyration-radius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
When comparing the mutants' simulations to the wildtype, R224W seems to show a bit more differences than C282S. Especially the whole protein's flexibility seems to be increased, but if this is a blessing or a curse is yet unkown. C282S would seem to be the less harmfull mutation of the two based on the MD results, yet it is known to be one of the more malicious ones.<br />
<br />
This task showed the power of molecular dynamics simulations, but it also showed its weakness. While it provides a lot of data one could easily be deluded by the results of a single run (as the differences between our two runs showed). In order to get reliable result multiple simulations should be performed to get some kind of average or consensus and to eliminate outliers.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28546Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:11:11Z<p>Joerdensv: /* Pymol analysis of average and bfactor */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
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Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Lambda 0<br />
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Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
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C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
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gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In this part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see (compare: <xr id="tab:avg_bfactor"/>) there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28545Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:10:17Z<p>Joerdensv: /* C-Alpha based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF (see <xr id="tab:rmsf_ca"/>) shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28544Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:09:43Z<p>Joerdensv: /* Protein based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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Item #frames Timestep (ps)<br />
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<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
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Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
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Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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<br />
gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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# Atoms 68603<br />
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Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
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Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar (see <xr id="tab:rmsf_prot"/>). The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28542Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:06:31Z<p>Joerdensv: /* Minimum distance between periodic boundary cells */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
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Item #frames Timestep (ps)<br />
Step 2001 5<br />
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<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
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T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
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<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
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Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
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<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
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# Atoms 68603<br />
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Box 2001 5<br />
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gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
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gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="minDistRun1"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of <xr id="minDistRun1"/>. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="minDistRun2"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in <xr id="minDistRun2"/>.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28541Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T19:03:29Z<p>Joerdensv: /* Calculation statistics */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
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gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
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C282S<br />
Checking file scwrl_C282S_md.xtc<br />
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gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
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gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
-----------------Run2<br />
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Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/1a6zC /mdrun_1a6zC/1a6zC_md.xtc<br />
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gcq#266: "Why Weren't You at My Funeral ?" (G. Groenhof)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _C282S/mdrun_scwrl_C282S/scwrl_C282S_md.xtc<br />
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gcq#106: "Count the Bubbles In Your Hair" (The Breeders)<br />
<br />
Checking file /mnt/home/student/bernhoferm/mstrprkt/task8/run2/models/scwrl _R224W/mdrun_scwrl_R224W/scwrl_R224W_md.xtc<br />
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gcq#281: "I'll Match Your DNA" (Red Hot Chili Peppers)<br />
<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28540Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T18:55:07Z<p>Joerdensv: /* Pymol analysis of average and bfactor */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the C1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the C1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With a script (provided by Dr. Marc Offman) we calculated the p-values for the RMS fluctuations to identify significant differences between the wildtype and the mutants. Surprisingly all three pairwise comparisons had very low p-values: <br />
<br />
* Wildtype and R224W: 8.079405e-57<br />
* Wildtype and C282S: 2.87453e-56<br />
* R224W and C282S: 5.45069e-25<br />
<br />
<br />
These results are to be considered with care though as the significance does not have to be caused by the mutations, but could also be caused by random events during the simulation. In fact, the wildtype alone (simulation 1 compared to simulation 2) showed up as highly significant (different).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed TODO?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<figtable id="tab:top_clusters"><br />
{| class="wikitable" style="float: left; margin: 1em 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 25:''' Comparison between the two most abundant clusters based on a cutoff of 0.18. The bigger cluster is colored green, the smaller red. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The two biggest clusters for each simulation are shown in <xr id="tab:top_clusters"/>. The clusters are aligned by the helices only which explains the good match for the MHC I domain, but it also helps to see the relative motions between both domains. The wildtype and C282S again are quite similar in that the both clusters have about the same structure. R224W in contrast shows quite strong inter-domain movement for the two most favored conformations. This further supports the suspected increased flexibility of the R224W mutant.<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_C282S_PP_Run2_Clusters.png&diff=28484File:Hemo MD C282S PP Run2 Clusters.png2012-08-31T17:43:20Z<p>Joerdensv: uploaded a new version of "File:Hemo MD C282S PP Run2 Clusters.png"</p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_R224W_PP_Run2_Clusters.png&diff=28483File:Hemo MD R224W PP Run2 Clusters.png2012-08-31T17:43:12Z<p>Joerdensv: uploaded a new version of "File:Hemo MD R224W PP Run2 Clusters.png"</p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_PP_Clusters.png&diff=28481File:Hemo MD 1a6zC PP Clusters.png2012-08-31T17:43:03Z<p>Joerdensv: uploaded a new version of "File:Hemo MD 1a6zC PP Clusters.png"</p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28478Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T17:39:25Z<p>Joerdensv: /* Internal RMSD */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Molecular dynamics simulations for HFE ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Trajectory visualization ===<br />
<br />
<br />
<figtable id="tab:traj_vis"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]<br />
| align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]<br />
| align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' Visualization of the molecular dynamics simulation trajectories. The calculated states (red) are superimposed on the PDB structure of 1a6zC (green). The timeframe of 10000ps was divided into 51 frames. The trajectories from left to right are: wildtype, R224W, and C282S.<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:traj_vis"/> shows the trajectories for the three MD simulations (WT, R224W, and C282S). The wildtype and R224W mostly maintain the starting structure, though some bigger parts are shifted as a whole. In both cases the upper and lower helices of the MHC I domain seem to move closer together and the M1 domain (right part in the figure) shifts around, but retains its general structure. The trajectory for C282S exhibits much less movement as the previous two which are quite flexible. Towards the end of the simulation there is a deformation of the M1 domain which is most likely caused by the missing disulfide bond due to the mutation. This deformation presumably prevents the later (but crucial) binding of beta-2-microglobulin to HFE.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_Clusters.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_Run2_Clusters.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_Run2_Clusters.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' The two most abundant clusters based on a cutoff of 0.18. The more often occuring is colored in green. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== Cluster analysis ===<br />
<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the whole protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:cluster_size_prot"/> and <xr id="tab:cluster_size_ca"/> show a clustering of the different structural states during the simulation. A cutoff of 0.18 was used for these clusters and they were separately calculated based on the whole protein (cf. <xr id="tab:cluster_size_prot"/>) and the C-alpha atoms (cf. <xr id="tab:cluster_size_ca"/>) only. As expected there are fewer clusters (conformations) for the C-alpha atoms only due to the exclusion of residue variations (rotations).<br />
<br />
The wildtype and C282S show a similar behavior in that the first two to three clusters represent the the majority conformations (the ones that are present the most during th simulation). There are 33 (14 C-alpha only) clusters for the wildtype and 25 (13 C-alpha only) clusters for C282S. R224W in contrast has 42 (20 C-alpha only) different clusters and the majority of the conformations is spread throughout more clusters. This suggests that there are not only more conformations, but they are also maintained for a longer time or assumed more often by the HFE mutant (i.e. it is more flexible).<br />
<br />
<br style="clear:both;"><br />
<br />
== Conclusion ==<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_R224W_PP_Run2_Clusters.png&diff=28475File:Hemo MD R224W PP Run2 Clusters.png2012-08-31T17:22:58Z<p>Joerdensv: </p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_R224W_MCB_Run2_Clusters.png&diff=28474File:Hemo MD R224W MCB Run2 Clusters.png2012-08-31T17:22:47Z<p>Joerdensv: </p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_C282S_PP_Run2_Clusters.png&diff=28473File:Hemo MD C282S PP Run2 Clusters.png2012-08-31T17:22:35Z<p>Joerdensv: </p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_C282S_MCB_Run2_Clusters.png&diff=28472File:Hemo MD C282S MCB Run2 Clusters.png2012-08-31T17:22:24Z<p>Joerdensv: </p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_PP_Clusters_View2.png&diff=28471File:Hemo MD 1a6zC PP Clusters View2.png2012-08-31T17:22:10Z<p>Joerdensv: </p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_PP_Clusters.png&diff=28470File:Hemo MD 1a6zC PP Clusters.png2012-08-31T17:21:57Z<p>Joerdensv: </p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_MCB_Clusters_View2.png&diff=28469File:Hemo MD 1a6zC MCB Clusters View2.png2012-08-31T17:21:38Z<p>Joerdensv: </p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_MCB_Clusters.png&diff=28468File:Hemo MD 1a6zC MCB Clusters.png2012-08-31T17:21:13Z<p>Joerdensv: </p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_C282S_MCB_cluster-sizes_Run2.png&diff=28452File:Hemo MD C282S MCB cluster-sizes Run2.png2012-08-31T16:35:10Z<p>Joerdensv: uploaded a new version of "File:Hemo MD C282S MCB cluster-sizes Run2.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_R224W_MCB_cluster-sizes_Run2.png&diff=28451File:Hemo MD R224W MCB cluster-sizes Run2.png2012-08-31T16:34:52Z<p>Joerdensv: uploaded a new version of "File:Hemo MD R224W MCB cluster-sizes Run2.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_MCB_cluster-sizes.png&diff=28450File:Hemo MD 1a6zC MCB cluster-sizes.png2012-08-31T16:34:29Z<p>Joerdensv: uploaded a new version of "File:Hemo MD 1a6zC MCB cluster-sizes.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_C282S_PP_cluster-sizes_Run2.png&diff=28448File:Hemo MD C282S PP cluster-sizes Run2.png2012-08-31T16:34:06Z<p>Joerdensv: uploaded a new version of "File:Hemo MD C282S PP cluster-sizes Run2.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_R224W_PP_cluster-sizes_Run2.png&diff=28445File:Hemo MD R224W PP cluster-sizes Run2.png2012-08-31T16:32:48Z<p>Joerdensv: uploaded a new version of "File:Hemo MD R224W PP cluster-sizes Run2.png"</p>
<hr />
<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_PP_cluster-sizes.png&diff=28444File:Hemo MD 1a6zC PP cluster-sizes.png2012-08-31T16:32:33Z<p>Joerdensv: uploaded a new version of "File:Hemo MD 1a6zC PP cluster-sizes.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=File:Hemo_MD_1a6zC_PP_cluster-sizes.png&diff=28443File:Hemo MD 1a6zC PP cluster-sizes.png2012-08-31T16:31:54Z<p>Joerdensv: uploaded a new version of "File:Hemo MD 1a6zC PP cluster-sizes.png"</p>
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<div></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28385Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T14:47:32Z<p>Joerdensv: /* Protein based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peak at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28374Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T13:56:31Z<p>Joerdensv: /* Dummy */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is described under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28361Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T13:33:24Z<p>Joerdensv: /* solvent accessible surface area */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
The preceding graphs show the Solvent accessible surface. As the hydrophilic, total and D Gsolv values are very similar we will just talk about the hydrophobic values.<br />
<br />
The most similar ones between those three are the wildtype and the R224W mutation which differ only in the strength of the fluctuation, of which the mutation has the higher one. Both fluctuate around about the same value. <br />
<br />
The less similar one is the C282S hydrophobic curve. It shows about the same fluctuation strength as the wildtype but in general a decrease towards the end can be seen. Therefore this might be an indicator, that the hydrophobic residues turn to the inside, but we cant be sure as there is no increase in the other values.<br />
<br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
In the area per residue averaged over the trajectory no clear difference can be seen.<br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' Ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The ramachandran plots (cf. <xr id="tab:ramachandran"/>) seem to follow the rules for the allowed and forbidden regions with a few exceptions. What is noticable though is that all of the allowed regions are almost filled to the maximum (i.e. no trend towards certain regions). Another difference are the regions above and below the left-handed alpha helix area (Psi: 110 to 180 and -180 to -150; Phi: 50 to 80) which are missing in the R224W mutation and almost non-existant in the C282S mutation. Though these areas have no significant structural element associated with them. Apart from that the plots appear almost the same, although they are hard to analyse as the dots are spread over a wide area and not clustered within distinct regions.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' RMSD matrices of the three calculated models over time (based on the whole protein) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' RMSD matrices of the three calculated models over time (based on the mainchain and C-betas) showing the RMSD between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Internal RMSD ===<br />
<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' RMSD of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' RMSD of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<xr id="tab:rmsd_vs_start"/> and <xr id="tab:rmsd_vs_avg"/> show the RMSD values for the different structural states compared to the starting and average structure respectively.<br />
<br />
The first (against starting structure) in particular reflects what could already be seen in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#RMSD_matrix|RMSD matrix]] section. The wildtype quickly assumes what seems to be its average structure and then periodically fluctuates around it. R224W slowly deviates from the starting structure until 6500ps where it has a moderate structural change and a strong one later at around 9500ps. C282S has a strong deviation at 2000ps and then remains quite stable till the end.<br />
<br />
When compared to the average structure the wildtype reaches its favored conformation at around 2000ps. There are some peaks around 4300ps, 5100ps, 7900ps, and 9100ps, but the RMSD alsways goes down afterwards. At the very end, though, is the highest peak which suggests a disruption of the structural equilibrium. This might be caused by the drop in the [[Molecular_Dynamics_Simulations_Analysis_Hemochromatosis#Minimum_distance_between_periodic_boundary_cells|MinDist]] between the boundaries below 2nm at the end of the simulation and therefore be a result of unwanted interactions between the simulated proteins.<br />
<br />
In contrast to the wildtype R224W has a higher RMSD to its average structure (0.3 compared to 0.2). This suggests a higher number of different states which are still quite similar to the starting structure (RMSD ~0.4). At the end of the simulation the equilibrium is disrupted (like in the wildtype simulation), but this time the reason is unkown as the minimum distance between boundaries is one of the highes values in this time period.<br />
<br />
Of the three simulations C282S shows the most stable average structure. It is accomplished by two major structural changes at around 2000ps and 3000ps. After that C282S exhibits the lowest RMSD fluctuations of the trio.<br />
<br />
<br style="clear:both;"><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28317Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:58:24Z<p>Joerdensv: /* Radius of gyration */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
These graphs show the radii of gyration in general als well as in each dimension. From start to end there is a slight increase in general gyrationradius for all models. However the fluctuation of the radius is the weakest for the wildtype and the C282S mutation. There seem to be most fluctuation in the R224W model. Also the R224W mutation has in general the highes radius of gyration.<br />
<br />
Another striking difference between the models is the low radius of gyration in the Z dimension for the mutations, whereas this type of gyration is in the wildtype only low for the first ~3000ps. In exchange both mutations gain more radius of gyration in the Y dimension compared to the wildtype.<br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The RMSD matrices for the whole protein (cf. <xr id="tab:rmsd_matrix_prot"/>) and the mainchain and C-beta atoms (cf. <xr id="tab:rmsd_matrix_mcb"/>) are almost identical. The C-beta matrices have slighty lower RMSD values which is not surprising as only a subset of the atoms is taken into consideration. The small difference between the two matrix groups suggests that most of the structural changes are based on backbone rearrangements and not on orientational changes (rotations) of the residues.<br />
<br />
The wildtype seems to periodically change between conformations as the RMSD goes up (green-yellow) and down (light blue) along the x-axis (time) for most of the different structural states (y-axis). The only noticable changes are in the at the beginning as the structure that is present in the first 1000ps seems to be quickly discarded (highest RMSD with the other states). Overall the changes are minor as the maximum RMSD is about 0.65 which is still considered quite low.<br />
<br />
The R224W mutations appears to be very stable in the beginning, but has two structural changes towards the end. A moderate one between 6500-8000ps and a strong one between 9500-10000ps. For the rest of the time it has even less structural fluctuations than the wildtype.<br />
<br />
The RMSD fluctuations for C282S exhibit an opposite behavior to R224W. There is a major structural change at 2000ps and some minor changes up to 3000ps. After 3000ps the structure remains almost the same until the end of the simulation. As the mutation is malign it can be assumed that it is a non-functional structure in which the proteins seems to be trapped.<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28313Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:37:48Z<p>Joerdensv: /* C-Alpha based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
The c-alpha based RMSF shows the same behavior as the whole-protein-based ones: the major differences are at around residue 20 and 220-235 of the R224W mutation.<br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28305Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:32:04Z<p>Joerdensv: /* C-Alpha based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28303Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:28:46Z<p>Joerdensv: /* Protein based */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In general the RMS fluctuations of all three models look similar. The most differing graph is the one of R224W which shows a major peak at residues 220-235 as well as only a small peat at around residue 20.<br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28298Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:03:35Z<p>Joerdensv: /* Total energy */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior with one exception: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps. The exception is the potential of the R224W mutation which is slightly higher than the other two models' potentials.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28294Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T11:00:01Z<p>Joerdensv: /* Potential */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average fluctuates around the same value for all three models themselves. However, these points differ slightly:<br />
<br />
The average potential of the wildtype and the C282S mutation tend to be around the same (~-9.195e+05) whereas the R224W mutation potential is slightly higher (~-9.19e+05).<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28292Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T10:51:43Z<p>Joerdensv: /* Minimum distance between periodic boundary cells */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average is all the time around the same value for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances can be seen in TABLETODO.<br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28291Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T10:48:28Z<p>Joerdensv: /* Protein-Protein */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average is all the time around the same value for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances were: <br />
<br style="clear:both;"><br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28290Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T10:47:36Z<p>Joerdensv: /* Protein-Water */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
<br />
<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average is all the time around the same value for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
The resulting minimal distances were: <br />
<br style="clear:both;"><br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== C-Alpha based ====<br />
<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
<br style="clear:both;"><br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.<br />
<br />
In case of the wildtype, both the number of hydrogen bonds as well as the number of pairs within 0.35nm are (compared to the rest) fairly low at first, rising in the first ~600ps. This may be an indication that at first a dense protein state is existent. Also from ~8000-8600ps there is a drop in the number of hydrogen bonds, whereas the number of pairs within 0.35nm does not show an equal behavior. Overall the numbers tend to be at the same level each after the first 600ps rise.<br />
<br />
The R224W model shows a different behavior over time:<br />
<br />
The first steps show a similar "both numbers low and rising till 600ps" behavior. But instead of having a fluctuation around one constant value there seems to be a slight decrease over time as well as bigger fluctuations. This affects both, the number of bonds as well as pairs within 0.35nm. <br />
<br />
<br />
The calculated model of the C282S mutation shows a different behavior at start than both preceding described, but a similar behavior to the R224W mutation:<br />
<br />
For the number of hydrogen bonds there are very high fluctuations at the first 1000ps (rising till 100ps, drop to 300ps, rise till 1000ps) with a slight overall decrease afterwards like for the R224W mutation. The number of pairs within 0.35nm seem to show a similar behavior, however because of the fluctuations this is not very clear.<br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28268Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T09:54:14Z<p>Joerdensv: /* Protein-Protein */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average is all the time around the same value for all three models.<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
The resulting minimal distances were: <br />
<br style="clear:both;"><br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
==== C-Alpha based ====<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The calculations show that in each of the three cases the number of bonds within the protein as well as their distances tend to stay the same.<br />
<br style="clear:both;"><br />
<br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensvhttps://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php?title=Molecular_Dynamics_Simulations_Analysis_Hemochromatosis&diff=28267Molecular Dynamics Simulations Analysis Hemochromatosis2012-08-31T09:46:50Z<p>Joerdensv: /* Pymol analysis of average and bfactor */</p>
<hr />
<div>[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Short task description ==<br />
<br />
<br />
Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]<br />
<br />
<br style="clear:both;"><br />
<br />
== Protocol ==<br />
<br />
<br />
A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].<br />
<br />
<br style="clear:both;"><br />
<br />
== Dummy ==<br />
Note: All pictures/graphs shown here are from the first run (in case of 1a6zC[wildtype]-pictures) or second run (in case of R224W- or C282S-mutation). The reason for this is depicted under LINKTOMINDISTTODO.<br />
<br />
<br />
=== Calculation statistics ===<br />
<figtable id="tab:simulation_stats"><br />
<caption>Statistics of the MD simulations</caption><br />
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed<br />
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| Wildtype<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| C282S<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years<br />
<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| R224W<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years<br />
<!--<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| L183P<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years<br />
|-<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0; font-style:italic;"| T217I<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day<br />
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years <br />
--><br />
|-<br />
|}<br />
</figtable><br />
<br />
GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.<br />
<!--<br />
GMXCheck:<br />
<br />
L183P<br />
Checking file scwrl_L183P_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68613<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)<br />
<br />
T217I<br />
Checking file scwrl_T217I_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68614<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#330: "Go back to the rock from under which you came" (Fiona Apple)<br />
<br />
Wildtype<br />
Checking file 1a6zC_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68607<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#134: "Push It Real Good" (Salt 'n' Pepa)<br />
<br />
C282S<br />
Checking file scwrl_C282S_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68603<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)<br />
<br />
R224W<br />
Checking file scwrl_R224W_md.xtc<br />
Reading frame 0 time 0.000<br />
# Atoms 68602<br />
Precision 0.001 (nm)<br />
Last frame 2000 time 10000.000<br />
<br />
<br />
Item #frames Timestep (ps)<br />
Step 2001 5<br />
Time 2001 5<br />
Lambda 0<br />
Coords 2001 5<br />
Velocities 0<br />
Forces 0<br />
Box 2001 5<br />
<br />
gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)<br />
<br />
--><br />
<br style="clear:both;"><br />
<br />
=== Energies ===<br />
==== Pressure ====<br />
<br />
<figtable id="tab:pressure"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' different pressures of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutatuin at position 282 (C282S)<br />
|}<br />
</figtable><br />
The plots in <xr id="tab:pressure"/> show the pressures of the calculated systems over time. These show that, although the pressures differ greatly in some cases, the average is still at about 0 (with minor fluctuations).<br />
<br style="clear:both;"><br />
<br />
==== Temperature ====<br />
<br />
<figtable id="tab:temperature"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 3:''' different temperature energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
The next thing we calculated were the temperatures. For all three models they can be seen in <xr id="tab:temperature"/>. The maximal deviation from the average is about 4 degrees for all models.<br />
<br style="clear:both;"><br />
<br />
==== Potential ====<br />
<br />
<figtable id="tab:potential"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 4:''' different potential energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
With gromacs we could also extract the potentials, as can be seen in <xr id="tab:potential"/>. As in the plots before the average is all the time around the same value for all three models.<br />
<br style="clear:both;"><br />
<br />
==== Total energy ====<br />
<br />
<figtable id="tab:total_energy"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 5:''' different total energies of the three calculated models over time. The red line denotes the average over 100 steps (500ps). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
In <xr id="tab:total_energy"/> the values of the total energies are denoted over the different states in time. Again we get an average with minor fluctuation at around the same value for each model.<br />
<br style="clear:both;"><br />
<br />
All these plots show the same behavior: average around the same value and look different between the three models. This can be expected as minor changes can introduce or eradicate bindings, therefore changing the overall energies which then influence all further steps.<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== Minimum distance between periodic boundary cells ===<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 6:''' different total energies of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
The first calculations for the mutations resulted in the minimum distances of TABLETODO. As there should be at least 2 nm distance in between at all time one can see that the mutations show the opposite. Therefore it might be possible that the protein affects itself which is not desired. To see if this states were calculated just by chance (random fluctuations that built up over time into an undesired direction) we repeated the calculations for all three models.<br />
<br style="clear:both;"><br />
<br />
<figtable id="comparison"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 7:''' different total energies of the three calculated models over time. The calculations are from the second run. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
The resulting minimal distances were: <br />
<br style="clear:both;"><br />
We therefore decided to use the model of the first calculation for the wildtype, and the models of the second calculation for the mutation types.<br />
<br />
=== RMSF for protein and C-alpha ===<br />
<br />
==== Protein based ====<br />
<figtable id="tab:rmsf_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 8:''' different RMS fluctuations (based on the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
==== C-Alpha based ====<br />
<figtable id="tab:rmsf_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 9:''' different RMS fluctuations (based on the the C-alpha atoms of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
==== Statistical values ====<br />
<!--<br />
<table id="tab:statistic_rmsf"><br />
{| class="wikitable" <br />
! <br />
! Average<br />
! StdDeviation<br />
! Ratio <br />
! significance<br />
|-<br />
! Wildtype<br />
! 0.183621323529412<br />
! 0.0605331780782099<br />
! 50.0281092824371<br />
! <br />
|-<br />
! R224W<br />
! 0.289421323529412<br />
! 0.111055982774513<br />
! 42.9806538073944<br />
! <br />
|-<br />
! C282S<br />
! 0.232049632352941<br />
! 0.0749374974847479<br />
! 51.0700344521995<br />
! <br />
|-<br />
<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' <br />
|}<br />
</table><br />
<br />
<br />
<br />
C282S Run2<br />
Variance: 0.0056156285292766<br />
Expectation: 0.232049632352941<br />
Standard Deviation: 0.0749374974847479<br />
Standard Error: 0.00454375319778049<br />
Ratio: 51.0700344521995<br />
Number of Entries: 272<br />
<br />
R224W Run2<br />
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg<br />
Variance: 0.012333431310013<br />
Expectation: 0.289421323529412<br />
Standard Deviation: 0.111055982774513<br />
Standard Error: 0.0067337580490602<br />
Ratio: 42.9806538073944<br />
Number of Entries: 272<br />
<br />
<br />
<br />
--------------Old Values-----------------<br />
|-<br />
! R224W<br />
! 0.232876838235294<br />
! 0.0895836329605749<br />
! 42.87282263833<br />
! <br />
|-<br />
! C282S<br />
! 0.268558823529412<br />
! 0.0982244780281883<br />
! 45.0924827835932<br />
! <br />
<br />
<br />
-----------------<br />
<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
8.079405e-57**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg<br />
2.87453e-56**<br />
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg<br />
5.45069e-25**<br />
--><br />
<br />
With the GIVENSCRIPTTODO we calculated the values for the t-Test:<br />
<br />
1a6zC to R224W :<br />
8.079405e-57**<br />
<br />
1a6zC to C282S :<br />
2.87453e-56**<br />
<br />
R224W to C282S :<br />
5.45069e-25**<br />
<br />
=== Pymol analysis of average and bfactor ===<br />
<br />
<br />
<figtable id="tab:avg_bfactor"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 11:''' Pictures of the model averages (average over MD calculated states) colored by the b-factor. The range is from blue (bfactor value beneath threshold [500]) to red (high b-factor values). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
In these part we evaluate the model averages and b-factors of each position. Because it is an average over all timesteps this averaged structure can be impossible in nature.<br />
<br />
As one can see there is a big change of b-factors when comparing the wildtype and both mutations we calculated. From both mutations the R224W one shows a bigger difference to the wildtype. As expected the positions "at the edges" <!-- most exposed?--> and those not in a secondary structure tend to have higher fluctuations/higher b-factors. It is worth noting, that even the beta sheets on the right side of the R224W picture (position 180 and higher) have (compared to wildtype and C282S mutation) pretty high b-factors. Also one can see that a little helix is inserted into the average structure of the R224W average model (right part of the picture, high b-factor [red], position ~220-225).<br />
<br style="clear:both;"><br />
<br />
=== Radius of gyration ===<br />
<br />
<figtable id="tab:gyration_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 12:''' different gyrations (based on the the C-alphas of the backbone of the protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<figtable id="tab:gyration_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 13:''' different gyrations (based on the the whole protein) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
<br />
=== solvent accessible surface area ===<br />
<br />
<figtable id="tab:sas"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 14:''' display of the different solvent accessible surface sizes of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<figtable id="tab:sas_res"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 15:''' display of the different solvent accessible surface sizes (normalized to per residue values) of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== hydrogen-bonds between protein and protein / protein and water ===<br />
==== Protein-Protein ====<br />
<br />
<figtable id="tab:hbonds_pp"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 16:''' the number of hydrogen bonds inside the protein of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br style="clear:both;"><br />
==== Protein-Water ====<br />
<br />
<figtable id="tab:hbonds_pw"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 17:''' the number of hydrogen bonds of the protein with water of the three calculated models over time. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br style="clear:both;"><br />
<br />
=== Ramachandran plots ===<br />
<br />
<figtable id="tab:ramachandran"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 18:''' ramachandran Plots of the three calculated models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== RMSD matrix ===<br />
<br />
<figtable id="tab:rmsd_matrix_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 19:''' rmsd matrices of the three calculated models over time (based on the whole protein) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<figtable id="tab:rmsd_matrix_mcb"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 20:''' rmsd matrices of the three calculated models over time (based on the mainchain and C-betas) showing the rmsd between two models. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== cluster analysis ===<br />
==== whole protein based ====<br />
<br />
<figtable id="tab:cluster_size_prot"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
<br />
| align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 21:''' Graphs showing the cluster sizes of the three models. The clustering was based on the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== C-alpha based ====<br />
<figtable id="cluster_size_ca"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]<br />
|-<br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
| align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 22:''' Graphs showing the cluster sizes of the three models. The clustering was based on the C-alpha atoms of the protein. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
=== internal RMSD ===<br />
<br />
==== against starting structure ====<br />
<figtable id="tab:rmsd_vs_start"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 23:''' rmsd of the calculated models over time against the beginning structure. From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
==== against average structure ====<br />
<figtable id="tab:rmsd_vs_avg"><br />
{| class="wikitable" style="float: right; margin: 0 0 0 1em; border: 2px solid darkgray;" cellpadding="0"<br />
<!--<br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
--><br />
! scope="row" align="left" |<br />
| align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
| align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px]]<br />
|-<br />
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 24:''' rmsd of the calculated models over time against the average structure (average based on all models over time). From left to right: 1a6zC (wildtype), mutation at position 224 (R224W) and mutation at position 282 (C282S)<br />
|}<br />
</figtable><br />
<br />
<br />
<br />
<br style="clear:both;"><br />
<br />
<!---<br />
Pymol analysis of average and bfactor <br />
Radius of gyration <br />
solvent accesible surface area <br />
hydrogen-bonds between protein and protein / protein and water <br />
Ramachandran plot: compare it to the standard ramachandran plots. also compare WT and mutations <br />
RMSD matrix <br />
cluster analysis <br />
internal RMSD <br />
--><br />
<br />
== References ==<br />
<br />
<br />
<references/><br />
<br />
<br style="clear:both;"></div>Joerdensv