View source for Molecular Dynamics Simulations Analysis Hemochromatosis

[[Hemochromatosis|Hemochromatosis]]>>[[Molecular Dynamics Simulations Analysis Hemochromatosis|Task 10: Molecular dynamics simulations analysis]]

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== Short task description ==


Detailed description: [[Task_10_-_Molecular_Dynamics_Simulations|Molecular dynamics simulations analysis]]

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== Protocol ==


A protocol with a description of the data acquisition and other scripts used for this task is available [[Task10 Hemochromatosis Protocol|here]].

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== Molecular dynamics simulations for HFE ==
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.


=== Calculation statistics ===
<figtable id="tab:simulation_stats">
<caption>Statistics of the MD simulations</caption>
{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Input
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. time
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| Calc. speed
! style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;"| time to reach 1 s

|-
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;  font-style:italic;"| Wildtype
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h31:15
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.750 ns/day
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 154350,8 years

|-
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;  font-style:italic;"| C282S
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:05
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.667 ns/day
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155075,9 years

|-
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;  font-style:italic;"| R224W
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h35:02
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.668 ns/day
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 155067,1 years
<!--
|-
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;  font-style:italic;"| L183P
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h24:04
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.909 ns/day
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152980,4 years
|-
| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;  font-style:italic;"| T217I
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 13h20:12
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 17.995 ns/day
| style="border-style: solid; padding-left:5px; padding-right:5px; text-align: right; border-width: 0 1px 1px 0;"| 152249,3 years 
-->
|-
|}
</figtable>

GMXcheck revealed for all calculations that all 2001 frames were calculated, resulting in a 10ns model.
<!--
GMXCheck:

L183P
Checking file scwrl_L183P_md.xtc
Reading frame       0 time    0.000
# Atoms  68613
Precision 0.001 (nm)
Last frame       2000 time 10000.000


Item        #frames Timestep (ps)
Step          2001    5
Time          2001    5
Lambda           0
Coords        2001    5
Velocities       0
Forces           0
Box           2001    5

gcq#11: "She's Not Bad, She's Just Genetically Mean" (Captain Beefheart)

T217I
Checking file scwrl_T217I_md.xtc
Reading frame       0 time    0.000
# Atoms  68614
Precision 0.001 (nm)
Last frame       2000 time 10000.000


Item        #frames Timestep (ps)
Step          2001    5
Time          2001    5
Lambda           0
Coords        2001    5
Velocities       0
Forces           0
Box           2001    5

gcq#330: "Go back to the rock from under which you came" (Fiona Apple)

Wildtype
Checking file 1a6zC_md.xtc
Reading frame       0 time    0.000
# Atoms  68607
Precision 0.001 (nm)
Last frame       2000 time 10000.000


Item        #frames Timestep (ps)
Step          2001    5
Time          2001    5
Lambda           0
Coords        2001    5
Velocities       0
Forces           0
Box           2001    5

gcq#134: "Push It Real Good" (Salt 'n' Pepa)

C282S
Checking file scwrl_C282S_md.xtc
Reading frame       0 time    0.000
# Atoms  68603
Precision 0.001 (nm)
Last frame       2000 time 10000.000


Item        #frames Timestep (ps)
Step          2001    5
Time          2001    5
Lambda           0
Coords        2001    5
Velocities       0
Forces           0
Box           2001    5

gcq#298: "Interfacing Space and Beyond..." (P. J. Harvey)

R224W
Checking file scwrl_R224W_md.xtc
Reading frame       0 time    0.000
# Atoms  68602
Precision 0.001 (nm)
Last frame       2000 time 10000.000


Item        #frames Timestep (ps)
Step          2001    5
Time          2001    5
Lambda           0
Coords        2001    5
Velocities       0
Forces           0
Box           2001    5

gcq#236: "Wait a Minute, aren't You.... ? (gunshots) Yeah." (Bodycount)

-->
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=== Trajectory visualization ===


<figtable id="comparison">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:hemo_MD_traj_1a6zC.gif|thumb|300px|Wildtype]]
|  align="right" | [[File:hemo_MD_traj_run2_R224W.gif|thumb|300px|R224W]]
|  align="right" | [[File:hemo_MD_traj_run2_C282S.gif|thumb|300px|C282S]]
|-
|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 2:''' TODO.
|}
</figtable>
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=== Energies ===


==== Pressure ====

<figtable id="tab:pressure">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_pressure.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_pressure_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_pressure_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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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).

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==== Temperature ====

<figtable id="tab:temperature">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_temperature.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_temperature_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_temperature_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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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.

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==== Potential ====

<figtable id="tab:potential">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_potential.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_potential_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_potential_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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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:

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).
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==== Total energy ====

<figtable id="tab:total_energy">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_totalEnergy.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_totalEnergy_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_totalEnergy_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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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.

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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.

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=== Minimum distance between periodic boundary cells ===


<figtable id="comparison">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo MD 1a6zC minPeriodicDist.png|thumb|300px]]
|  align="right" | [[File:Hemo MD R224W minPeriodicDist.png|thumb|300px]]
|  align="right" | [[File:Hemo MD C282S minPeriodicDist.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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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.

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<figtable id="comparison">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo MD 1a6zC minPeriodicDist_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo MD R224W minPeriodicDist_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo MD C282S minPeriodicDist_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
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The resulting minimal distances can be seen in TABLETODO.
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.

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=== RMSF for protein and C-alpha  ===

==== Protein based ====

<figtable id="tab:rmsf_prot">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_prot_rmsf.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_prot_rmsf_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_prot_rmsf_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

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.

==== C-Alpha based ====

<figtable id="tab:rmsf_ca">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_ca_rmsf.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_ca_rmsf_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_ca_rmsf_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>

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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.

==== Statistical values ====
<!--
<table id="tab:statistic_rmsf">
{|  class="wikitable" 
! 
! Average
! StdDeviation
! Ratio 
! significance
|-
! Wildtype
! 0.183621323529412
! 0.0605331780782099
! 50.0281092824371
! 
|-
! R224W
! 0.289421323529412
! 0.111055982774513
! 42.9806538073944
! 
|-
! C282S
! 0.232049632352941
! 0.0749374974847479
! 51.0700344521995
! 
|-

|+ style="caption-side: bottom; text-align: left" |<font size=1>'''Table 10:''' 
|}
</table>



C282S Run2
Variance: 0.0056156285292766
Expectation: 0.232049632352941
Standard Deviation: 0.0749374974847479
Standard Error: 0.00454375319778049
Ratio: 51.0700344521995
Number of Entries: 272

R224W Run2
perl calcVar.pl Files/Run2_R224W.prot.rmsf.xvg
Variance: 0.012333431310013
Expectation: 0.289421323529412
Standard Deviation: 0.111055982774513
Standard Error: 0.0067337580490602
Ratio: 42.9806538073944
Number of Entries: 272



--------------Old Values-----------------
|-
! R224W
! 0.232876838235294
! 0.0895836329605749
! 42.87282263833
! 
|-
! C282S
! 0.268558823529412
! 0.0982244780281883
! 45.0924827835932
! 


-----------------

joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg
8.079405e-57**
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/1a6zC.prot.rmsf.xvg Files/Run2_C282S.prot.rmsf.xvg
2.87453e-56**
joerdensv@i12k-biolab01:~/Desktop/MastPrakt/Task10$ perl NewVarCalc.pl Files/Run2_C282S.prot.rmsf.xvg Files/Run2_R224W.prot.rmsf.xvg
5.45069e-25**
-->

With the GIVENSCRIPTTODO we calculated the values for the t-Test:

1a6zC to R224W :
8.079405e-57**

1a6zC to C282S :
2.87453e-56**

R224W to C282S :
5.45069e-25**

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=== Pymol analysis of average and bfactor ===


<figtable id="tab:avg_bfactor">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zCProtAvg.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224WProtAvg_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282SProtAvg_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

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.

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 style="clear:both;">

=== Radius of gyration ===

<figtable id="tab:gyration_ca">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_ca_gyration.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_ca_gyration_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_ca_gyration_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

<figtable id="tab:gyration_prot">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_prot_gyration.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_prot_gyration_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_prot_gyration_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>

<br style="clear:both;">
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.

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 style="clear:both;">

=== solvent accessible surface area ===

<figtable id="tab:sas">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_SAS.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_SAS_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_SAS_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">
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.

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. 

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.


<figtable id="tab:sas_res">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_resSAS.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_resSAS_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_resSAS_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>

<br style="clear:both;">
In the area per residue averaged over the trajectory no clear difference can be seen.

<br style="clear:both;">

=== hydrogen-bonds between protein and protein / protein and water ===
==== Protein-Protein ====

<figtable id="tab:hbonds_pp">
{|  class="wikitable"  style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_hBondsP2P.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_hBondsP2P_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_hBondsP2P_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>

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 style="clear:both;">

==== Protein-Water ====

<figtable id="tab:hbonds_pw">
{|  class="wikitable"  style="float: left; margin: 0 0 0 1em; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_hBondsP2W.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_hBondsP2W_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_hBondsP2W_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>

Although the inner-protein bonds seem to stay the same (see PREVIOUSSECTIONTODO), the bonds formed with hydrogen show a different behavior.

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.

The R224W model shows a different behavior over time:

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. 


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:

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 style="clear:both;">

=== Ramachandran plots ===

<figtable id="tab:ramachandran">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_ramaPlot.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_ramaPlot_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_ramaPlot_Run2.png|thumb|300px|C282S]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

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 style="clear:both;">

=== RMSD matrix ===

<figtable id="tab:rmsd_matrix_prot">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_PP_rmsd-matrix.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_PP_rmsd-matrix_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_PP_rmsd-matrix_Run2.png|thumb|300px|C282S]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

<figtable id="tab:rmsd_matrix_mcb">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_MCB_rmsd-matrix.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_MCB_rmsd-matrix_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_MCB_rmsd-matrix_Run2.png|thumb|300px|C282S]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

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.

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.

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.

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 style="clear:both;">

=== cluster analysis ===

==== whole protein based ====

<figtable id="tab:cluster_size_prot">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
<!--Mit reinnehmen? Unklar was man sagen könnt [Vadim]
|  align="right" | [[File:Hemo_MD_1a6zC_PP cluster-id-over-time.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_PP cluster-id-over-time_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_PP cluster-id-over-time_Run2.png|thumb|300px]]
|-

|  align="right" | [[File:Hemo_MD_1a6zC_PPcluster-transitions.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_PP cluster-transitions_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_PP cluster-transitions_Run2.png|thumb|300px]]
|-
-->
|  align="right" | [[File:Hemo_MD_1a6zC_PP cluster-sizes.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_PP cluster-sizes_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_PP cluster-sizes_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">


<br style="clear:both;">

==== C-alpha based ====

<figtable id="cluster_size_ca">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
<!-- Mit reinnehmen? Unklar was man sagen könnt [Vadim]
|  align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-id-over-time.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_MCB cluster-id-over-time_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_MCB cluster-id-over-time_Run2.png|thumb|300px]]
|-
|  align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-transitions.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_MCB cluster-transitions_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_MCB cluster-transitions_Run2.png|thumb|300px]]
|-
-->
|  align="right" | [[File:Hemo_MD_1a6zC_MCB cluster-sizes.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_R224W_MCB cluster-sizes_Run2.png|thumb|300px]]
|  align="right" | [[File:Hemo_MD_C282S_MCB cluster-sizes_Run2.png|thumb|300px]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">


<br style="clear:both;">

=== Internal RMSD ===


<figtable id="tab:rmsd_vs_start">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
<!--
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-start.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-start_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-start_Run2.png|thumb|300px|C282S]]
|-
-->
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_rmsd-allProt-atom-vs-start.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_rmsd-allProt-atom-vs-start_Run2.png|thumb|300px|C282S]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

<figtable id="tab:rmsd_vs_avg">
{|  class="wikitable"  style="float: left; margin: 0 0 1em 0; border: 2px solid darkgray;"   cellpadding="0"
<!--
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_rmsd-ca-vs-average.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_rmsd-ca-vs-average_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_rmsd-ca-vs-average_Run2.png|thumb|300px|C282S]]
|-
-->
!  scope="row" align="left" |
|  align="right" | [[File:Hemo_MD_1a6zC_rmsd-all-atom-vs-average.png|thumb|300px|Wildtype]]
|  align="right" | [[File:Hemo_MD_R224W_rmsd-all-atom-vs-average_Run2.png|thumb|300px|R224W]]
|  align="right" | [[File:Hemo_MD_C282S_rmsd-all-atom-vs-average_Run2.png|thumb|300px|C282S]]
|-
|+ 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)
|}
</figtable>
<br style="clear:both;">

<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.

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.

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.

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.

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 style="clear:both;">

== References ==


<references/>

<br style="clear:both;">