Difference between revisions of "Task 10: Molecular Dynamics Analysis"

In this task we are going to analyze the results of the molecular dynamics simulations of task 8. A detailed task description can be found here. The analysis focuses on this tutorial.

Native

A BRIEF CHECK OF RESULTS

In order to verify that our simulation ran successfully we used the command line tool gmxcheck. we executed it as follows for our .xtc file:

``` ```

```gmxcheck -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc ```

How many frames are in the trajectory file and what is the time resolution?

We observed 2001 frames with a time resolution of 5ps.

How long did the simulation run in real time (hours), what was the simulation speed (ns/day) and how many years would the simulation take to reach a second?

The simulation ran 4h00:39 and had a simulation speed of 59.835 ns/day. To calculate 1 second we would need (1 / (59 * (10^(-9)))) / 365 = 46 436.0344 years

Which contribution to the potential energy accounts for most of the calculations?

• potential energy: -4.57312e+05 kJ/mol

VISUALIZATION OF RESULTS

We extracted 1000 frames from the trajectory (-dt 10), leaving out the water (selected Protein when asked for a selection). Moreover, we will remove the jumps over the boundaries and make a continuous trajectory (-pbc nojump):

``` trjconv -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc -o protein.pdb -pbc nojump -dt 10 ```

After that we opened the generated protein.pdb file with pymol. Here we changed the coloring to spectrum by typing the following to the pymol command line:

``` ```

```spectrum ```

In a next step we enabled the the visualization of the cell with this command:

``` ```

```show cell ```

In order to remove the tumbeling and wiggeling motion of our protein we used the command intra_fit since we are only interested in the internal motions of the protein:

``` ```

```intra_fit protein ```

The results of these actions can be seen in figure 1 and 2. Figure one shows our WT protein in line view which makes it able to see the motion of the side chains. Figure to shows the protein in cartoon view to see the overall movement of the secondary structure elements.

 Figure 1: motion of protein in line view Figure 2: motion of protein in cartoon view

QUALITY ASSURANCE

CONVERGENCE OF ENERGY TERMS

In this part of quality assurance we analyzed different metrics of our MD simulation by creating plots from our *.edr file. We created plots for the temperature, pressure, energy, volume, density, box, coulomb and van der waals values of our MD simulation by using the tool g_energy as follows:

``` ```

```//calculating temperature enter then "12 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/temperature.xvg ``````//calculating pressure enter then "13 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/pressure.xvg ``````//calculating energy (potential, kinetic and total) enter then "9\n10\n11 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/energy.xvg ``````//calculating volume enter then "18 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/volume.xvg ``````//calculating density enter then "19 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/density.xvg ``````//calculating box enter then "15\n16\n17 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/box.xvg ``````//calculating coulomb enter then "48\n50 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/coulomb-inter.xvg ``````//calculating van der waals enter then "49\n51 0" g_energy -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.edr -o ../../../Dropbox/Studium/3_semester/master_praktikum/task10/vanderwaals-inter.xvg ```

Temperature Over Time
 Figure 3: Fluctuation of temperature over time in WT

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Temperature" 297.886 0.007 1.57824 "-0.0105059 (K)"

In figure 3 we can see that the temperature stays quite the same over the whole simulation which might be interpreted as that our system has reached its stable temperature for simulation.

Pressure over Time
 Figure 4: Fluctuation of pressure over time in WT
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Pressure" 1.02313 0.023 133.659 "-0.0928554 (bar)"
Energy over Time
 Figure 5: Potential, kinetic and total energy over time in WT
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Potential" -457312 61 640.502 " -423.792 (kJ/mol)" "Kinetic En." 81855.1 1.9 433.677 " -2.88684 (kJ/mol)" "Total Energy" -375457 61 784.246 " -426.678 (kJ/mol)"
Volume over Time
 Figure 6: Fluctuation of volume over time in WT

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Volume" 356.629 0.041 0.397685 " -0.151337 (nm^3)"
Density over Time
 Figure 7: Fluctuation of density over time in WT

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Density" 1021.28 0.12 1.13884 "0.433467 (kg/m^3)"
Box over Time
 Figure 8: box size over time in WT
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Box-X" 7.95996 " 0.0003" 0.00295875 "-0.00112603 (nm)" "Box-Y" 7.95996 " 0.0003" 0.00295875 "-0.00112603 (nm)" "Box-Z" 5.62854 0.00021 0.00209216 "-0.000796226 (nm)"
Coulomb over Time
 Figure 9: Fluctuation of coulomb energies over time in WT
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Coul-SR:Protein-non-Protein" " -20429.9" "110" "425.888" "-561.713 (kJ/mol)" "Coul-14:Protein-non-Protein" " 0" " 0" " 0" " 0 (kJ/mol)"
Van der Waals over Time
 Figure 10: Fluctuation of van der waals energies over time in WT

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "LJ-SR:Protein-non-Protein" "-2172.28" "15" "136.956" "-103.12 (kJ/mol)" "LJ-14:Protein-non-Protein" " 0" " 0" " 0" " 0 (kJ/mol)"

Questions

1. What is the average temperature and what is the heat capacity of the system?

The average temperature of our system is 297.886 K and the heat capacity ranges from 303 to 292. (values taken from figure 3).

2. What are the terms plotted in the files energy.xvg and box.xvg

In energy.xvg we plot the energy values of kinetic energy, potential energy and total energy over the time of our simulation.

In box.xvg we plot the size of the box around our protein which is given in nm.

3. Estimate the plateau values for the pressure, the volume and the density.

We consider the plateau value as two values, the upper and lower plateau. These plateaus represent the maximum and minimum values of our given plots.

Pressure:

• upper plateau:450 bar
• lower plateau: -450 bar

Volume:

• upper plateau: 357.9 nm^3
• lower plateau: 355.8 nm^3

Density:

• upper plateau: 1026 kg/m^3
• lower plateau: 1017 kg/m^3

4. What are the terms plotted in the files coulomb-inter.xvg and vanderwaals-inter.xvg

The coloumb and van der waals energys of our WT over time.

MINIMUM DISTANCES BETWEEN PERIODIC IMAGES

We ran gromacs with the following command:

``` ```

```//when asked for group selection we selected group 1 g_mindist -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -od /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/minimal-periodic-distance.xvg -pi ```

And produced the plot seen in figure 11.

 Figure 11: minimum periodic distance on the protein over time in WT

In a next step we produced a plot which only uses the C-alpha atoms to calculate the minimum distance between periodic images. For this we used the same command as before. Although, we selected group 3 this time. The result can be seen in figure 12.

 Figure 12: minimum periodic distance on C-alpha atoms over time in WT

Questions

What was the minimal distance between periodic images and at what time did that occur?

The shortest periodic distance is 1.73442 (nm) at time 2720 (ps), between atoms 563 and 5314.

What happens if the minimal distance becomes shorter than the cut-off distance used for electrostatic interactions? Is it the case in your simulations?

If the distance would become shorter than the cut-off distance used for electrostatic interactions our energy would dramatically increase. This did not happen for our simulation so we can assume that our measured value of 1.73 nm is still higher than the cutoff.

Run now g_mindist on the C-alpha group, does it change the results? What does is mean for your system?

With C-alpha group we got a shortest periodic distance of 2.4249 (nm) at time 6805 (ps), between atoms 589 and 5297. This is an increase of the distance since we only consider C-alpha atoms and no side chains as in our previous calculation.

ROOT MEAN SQUARE FLUCTUATIONS

In this part of the analysis we are going to have a look at the root mean square fluctuations. By analyzing this value for our structure we might be able to figure out which parts of our protein are more flexible than others.

To do so we executed the following command:

``` ```

```g_rmsf -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsf-per-residue.xvg -ox /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/average.pdb -oq /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/bfactors.pdb -res ```

Which gives us three result files:

• rmsf-per-residue.xvg : this file contains a plot over the whole structure length of the RMSF values
• average.pdb : is the average conformation of the structure
• bfactors.pdb: assigned b factor values to the residues which gives us information about the flexibility
 Figure 13: RMSF per residue in WT Figure 14: B-factor in WT, front view Figure 15: B-factor in WT, back view

Questions

Indicate the start and end residue for the most flexible regions and the maximum amplitudes.

The most flexible region in our structure is from position 131 to 152 which is a loop connecting two alpha-helices. This can be observed in the plot in figure 13 as well as in the 3D view of our protein in figure 14. In general we can say that most of the parts are rather rigid, especially the residues which are located within the core. We observe slightly more flexibility in some alpha-helices which are located at the surface of the protein. Such as the residues located at position 197, 208, 215 and 216. There are also some beta-sheets which contain slightly flexible residues such as residues at position 413 and 420.

CONVERGENCE OF RMSD

It is adviced in the tutorial to remove the jumps of our protein and bring it back to the middle. This is done with the following command:

``` ```

```trjconv -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc -o 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -pbc nojump ```

Then we calculated the RMSD for the whole protein and a second time only for the backbone with the following command:

``` ```

```//whole protein: select 1 and 1 again g_rms -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-all-atom-vs-start.xvg ``````//backbone only: select 4 and 4 again g_rms -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-backbone-vs-start.xvg ```

After that we calculated the RMSD for the average protein structure with the following commands, once for the whole protein and once for the backbone only:

``` ```

```trjconv -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/protein.xtc `````` ``````g_rms -f /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/protein.xtc -s /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/average.pdb -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-all-atom-vs-average.xvg ``````g_rms -f /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/protein.xtc -s /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/average.pdb -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-backbone-vs-average.xvg ```

We received then the following results:

 Figure 16: RMSD all atom vs start in WT Figure 17: RMSD backbone vs start in WT
 Figure 18: RMSD all atom vs average in WT Figure 19: RMSD backbone vs average in WT

Questions

If observed, at what time and value does the RMSD reach a plateau?

We observed in the figures (16-17, against start structure) that the plateau is reached after 5000ps with the following approximated values:

• All atom vs start: 0.225
• Backbone vs start: 0.175

For the average structures we observed that the plateau is reached earlier after 300ps with the following approximated values:

• All atom vs average: 0.16
• Backbone vs average: 0.125

Briefly discuss two differences between the graphs against the starting structure and against the average structure. Which is a better measure for convergence?

As we can see the plateau is reached earlier in the average structure than in the start structure. The reason for this might be that the average structure is closer to the equilibrium than the start structure. Hence, also the RMSD values are lower here.

Since the average structures converges earlier than the starting structure we think that this plots are the better ones to judge whether convergence has been reached or not.

To calculate the radius of gyration we executed the following command:

``` ```

```g_gyrate -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/radius-of-gyration.xvg ```

And received the following plot :

 Figure 20: Radius of gyration in WT

Questions

At what time and value does the radius of gyration converge?

The radius of gyration fluctuates between 1.90 to 1.95 nm. The value for RGz is fluctuating between 1.55 to 1.70, RGx is fluctuating between 1.45 to 1.70 and RGy is fluctuating between 1.45 to 1.55.

STRUCTURAL ANALYSIS: PROPERTIES DERIVED FROM CONFIGURATIONS

SOLVENT ACCESSIBLE SURFACE AREA

In this part of our analysis we are checking the solvent accessible area of our protein over time. To calculate these values we executed the following command:

``` ```

```// select 1 and 1 again when asked for group g_sas -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/solvent-accessible-surface.xvg -oa /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/atomic-sas.xvg -or /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/residue-sas.xvg ```

We received the following 3 output plots:

 Figure XX solvent accessible area over time in WT Figure XX solvent accessible area per atom in WT Figure XX solvent accessible area per residue in WT

HYDROGEN BONDS

In this part of our analysis we are looking at inter H-bonds within our protein and at H-bonds from our protein to the surrounding solvent. To do so we executed the following command twice:

``` ```

```// first time with 1 and 1 again to calculate inter H-bonds // second time with 1 and 12 to calculate H-bonds from the protein to the surrounding solvent g_hbond -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -num /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/hydrogen-bonds-protein-water.xvg ```

We received the following results seen in the figures below:

 Figure XX: Number of intra H-bonds over time in WT Figure XX: Number of protein to solvent H-bonds over time in WT

Questions

Discuss the relation between the number of hydrogen bonds for both cases and the fluctuations in each.

RAMACHANDRAN (PHI/PSI) PLOTS

In this part of the analysis we have a look at the phi/psi angles in our protein. To do the calculations we executed the following command:

``` ```

```g_rama -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/ramachandran.xvg ```

 Figure XX: ramachandran plot in WT

ANALYSIS OF DYNAMICS AND TIME-AVERAGED PROPERTIES

ROOT MEAN SQUARE DEVIATIONS AGAIN

In this part of the analysis we calculate the RMSD of our structure versus our structure over time. To do so we executed the following two commands:

``` ```

```//RMSD calculation g_rms -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -f2 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -m rmsd-matrix.xpm -dt 10 ``````//coloring of plot xpm2ps -f rmsd-matrix.xpm -o rmsd-matrix.eps -rainbow blue ```

Finally we received the following plot:

 Figure XX: RMSD of WT structure vs WT structure

Questions

What is interesting by choosing the group "Mainchain+Cb" for this analysis? Think about the different proteins used for this practical.

How many transitions do you see?

What can you conclude from this analysis? Could you expect such a result, justify?

Cluster Analysis

``` ```

```//when asked for input select 6 and 6 again g_cluster -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -dm /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-matrix.xpm -dist /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/rmsd-distribution.xvg -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/clusters.xpm -sz /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/cluster-sizes.xvg -tr /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/cluster-transitions.xpm -ntr /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/cluster-transitions.xvg -clid /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/cluster-id-over-time.xvg -cl /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/clusters.pdb -cutoff 0.1 -method gromos -dt 10 ```

0.1 Cutoff
 Figure XX: RMSD distribution with a cutoff of 0.1 in WT Figure XX: Cluster size with a cutoff of 0.1 in WT Figure XX: Cluster transition with a cutoff of 0.1 in WT Figure XX: Cluster id over time with a cutoff of 0.1 in WT

0.13 Cutoff
 Figure XX: RMSD distribution with a cutoff of 0.13 in WT Figure XX: Cluster size with a cutoff of 0.13 in WT Figure XX: Cluster transition with a cutoff of 0.13 in WT Figure XX: Cluster id over time with a cutoff of 0.13 in WT

DISTANCE RMSD

``` ```

```//select 1 and 1 again when asked for input g_rmsdist -s 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md.tpr -f 1J8U_nosol_after_SCWRL_no_h_merged_crystal_water_md_nojump.xtc -o /home/student/Dropbox/Studium/3_semester/master_praktikum/task10/distance-rmsd.xvg ```

 Figure XX: distance RMSD over time in WT

P281L

A BRIEF CHECK OF RESULTS

In order to verify that our simulation ran successfully we used the command line tool gmxcheck. we executed it as follows for our .xtc file:

``` ```

```gmxcheck -f P281L_md.xtc ```

How many frames are in the trajectory file and what is the time resolution?

We observed 2001 frames with a time resolution of 5ps.

How long did the simulation run in real time (hours), what was the simulation speed (ns/day) and how many years would the simulation take to reach a second?

The simulation ran 3h14 and had a simulation speed of 73.133 ns/day. To calculate 1 second we would need 0.328*10^9/(365*24) = 37442.92 years

Which contribution to the potential energy accounts for most of the calculations?

• potential energy: -4.57346e+05 kJ/mol
• greatest contribution by Coulomb: -4.45954e+05 kJ/mol

VISUALIZATION OF RESULTS

We extracted 1000 frames from the trajectory (-dt 10), leaving out the water (selected Protein when asked for a selection). Moreover, we will remove the jumps over the boundaries and make a continuous trajectory (-pbc nojump):

``` trjconv -s P281L_md.tpr -f P281L_md.xtc -o protein.pdb -pbc nojump -dt 10 ```

After that we opened the generated protein.pdb file with pymol.

QUALITY ASSURANCE

CONVERGENCE OF ENERGY TERMS

In this part of quality assurance we analyze different metrics of our MD simulation by creating plots from our *.edr file. We created plots for for pressure, temperature, potential and total energy of our MD simulation.

Temperature Over Time
 Figure XX: Fluctuation of temperature over time in P281L

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Temperature" 297.887 0.0054 1.58026 0.0204823

In figure XX we can see that the temperature stays quite the same over the whole simulation. The average temperature is the same as in the WT simulation, the RMSD is slightly higher. That means we have more or greater outliers. Regarding the plot, we suggest that there are more outliers. There is especially one at about 8500 ps with a very high temperature. Such a high temperature could not be seen in the WT simulation. But there are only few differences, therefore we suggest, that the differences are not due to our mutation but that the differences are caused by the non-deterministic simulation process. The temperature has reached some kind of convergence. Therefore the simulation was probably successful.

Pressure over Time
 Figure XX: Fluctuation of pressure over time in P281L
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Pressure" 1.01427 0.011 133.436 -0.0559964

Like in the analysis of the pressure, there seems to be no dramatic change compared to the WT simulation. It seems, that the pressure also converges in the simulation of the mutation. Therefore the simulation was probably successful.

Energies over Time

Total Energy

 Figure XX: Total energy over time in P281L
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Total Energy" -375514 53 783.088 -307.507

Potential Energy

 Figure XX: Potential energy over time in P281L
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Total Energy" -457346 54 638.456 -313.132

In the potential and the total energy statistic for the mutation, we can observe a decrease in the energies for the first 4000 ps. Afterwards the energy converges. Therefore we assume that the simulation of the mutation was successful.

MINIMUM DISTANCES BETWEEN PERIODIC IMAGES

 Figure XX: minimum periodic distance on the protein over time in P281L
 Figure XX: minimum periodic distance on C-alpha atoms over time in P281L

We check the shortest periodic distances, because we want to avoid interactions between a atom to itself in two periodic images. Such an unphysical interaction would dramatically influence the simulation. The result would be nonsense.

The shortest periodic distance in the whole protein is 1.9083 (nm) at time 55 (ps), between atoms 1198 and 5323. The shortest shortest periodic distance between two C-alpha is 2.75268 nm at time 2320 ps, between atoms 2715 and 5306.

Therefore we suggest, that no such unphysical interaction occured in the simulation.

ROOT MEAN SQUARE FLUCTUATIONS

In this part of the analysis we are going to have a look at the root mean square fluctuations. By analyzing this value for our structure we might be able to figure out which parts of our protein are more flexible than others.

 Figure XX: RMSF per residue in P281L Figure XX: RMSF of the backbone per residue Figure XX: B-factor in P281L

Compared to the WT simulation the peak at about residue 45 in the RMSF per residue plot decreased from 0.45 to 0.275 RMSF. This might happen due to the non-deterministic simulation process, but the rest of the plot seems to be untouched. Therefore we assume it could be an effect of the mutation. Due to the similarity to the WT simulation and our decision about its quality, we can call the mutation simulation also successful.

 Figure XX: Gyration in P281L

The plots for the gyration of the WT and of the mutation are quite different. But this might be due to the "non-deterministic" simulation process. But this is not essential for the quality assessment. The overall gyration radius is quite stable at 1.94 nm. The gyration radius of the three axis are converging. RgX and RgY are quite overlapping. Wheras RgZ converges with the RgX and RgY at about 1.4 nm. Therefore we can assume, that the simulation was successful.

STRUCTURAL ANALYSIS: PROPERTIES DERIVED FROM CONFIGURATIONS

SOLVENT ACCESSIBLE SURFACE AREA

 Figure XX: solvent accessible area over time in P281L Figure XX: solvent accessible area per atom in P281L Figure XX: solvent accessible area per residue in P281L

There seems to be no difference to the WT simulation.

HYDROGEN BONDS

In this part of our analysis we are looking at inter H-bonds within our protein and at H-bonds from our protein to the surrounding solvent.

 Figure XX: intra protein hydrogen bonds in P281L Figure XX: hydrogen bonds between solvent and protein in P281L

The hydrogen bonds within the protein seems to be untouched by the mutation. Whereas the hydrogen bonds of the protein to the water show a significant difference. In the WT there is a increase in hydrogen bonds from 5500 ps to 8000 ps. This "wave" is missing in the plot of the mutation's simulation. Perhaps this is just a result of the non-deterministic simulation process. Therefore it is probably caused by the mutation.

RMSD matrix

 Figure XX: rmsd matrix of P281L Figure XX: rmsd distribution of P281L

Cluster analysis

 Figure XX: clusters of P281L Figure XX: cluster ids over time of P281L Figure XX: cluster sizes of P281L Figure XX: cluster transitions of P281L

Distance RMSD

 Figure XX: distance RMSD of P281L

R408W

A BRIEF CHECK OF RESULTS

In order to verify that our simulation ran successfully we used the command line tool gmxcheck. we executed it as follows for our .xtc file:

``` ```

```gmxcheck -f R408W_md.xtc ```

How many frames are in the trajectory file and what is the time resolution?

We observed 2001 frames with a time resolution of 5ps.

How long did the simulation run in real time (hours), what was the simulation speed (ns/day) and how many years would the simulation take to reach a second?

The simulation ran 4h07 and had a simulation speed of 76.846 ns/day. To calculate 1 second we would need 0.312*10^9/(365*24)= 35616.43 years

Which contribution to the potential energy accounts for most of the calculations?

• potential energy: -4.57166e+05 kJ/mol
• greatest contribution by Coulomb: -4.46176e+05 kJ/mol

VISUALIZATION OF RESULTS

We extracted 1000 frames from the trajectory (-dt 10), leaving out the water (selected Protein when asked for a selection). Moreover, we will remove the jumps over the boundaries and make a continuous trajectory (-pbc nojump):

``` trjconv -s R408W_md.tpr -f R408W_md.xtc -o protein.pdb -pbc nojump -dt 10 ```

After that we opened the generated protein.pdb file with pymol.

QUALITY ASSURANCE

CONVERGENCE OF ENERGY TERMS

In this part of quality assurance we analyze different metrics of our MD simulation by creating plots from our *.edr file. We created plots for for pressure, temperature, potential and total energy of our MD simulation.

Temperature Over Time
 Figure XX: Fluctuation of temperature over time in R408W

 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" Temperature 297.883 0.008 1.57981 -0.00616634

In figure 3 we can see that the temperature stays quite the same over the whole simulation which might be interpreted as that our system has reached its stable temperature for simulation.

Pressure over Time
 Figure XX: Fluctuation of pressure over time in R408W
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Pressure" 1.0242 0.03 133.452 -0.0978398
Total Energy over Time
 Figure XX: Total energy over time in R408W
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Total Energy" -375304 46 779.24 -291.686
Potential Energy over Time
 Figure XX: Potential energy over time in R408W
 "Energy" "Average" "Err.Est." "RMSD" "Tot-Drift" "Potential Energy" -457166 46 634.117 -289.993

MINIMUM DISTANCES BETWEEN PERIODIC IMAGES

We ran gromacs with the following command:

``` ```

```//when asked for group selection we selected group 1 g_mindist -f R408W_md.xtc -s R408W_md.tpr -od minimal-periodic-distance.xvg -pi ```

And produced the plot seen in figure 11.

 Figure XX: minimum periodic distance on the protein over time in R408W

In a next step we produced a plot which only uses the C-alpha atoms to calculate the minimum distance between periodic images. For this we used the same command as before. Although, we selected group 3 this time. The result can be seen in figure 12.

 Figure XX: minimum periodic distance on C-alpha atoms over time in R408W

Questions

What was the minimal distance between periodic images and at what time did that occur?

The shortest periodic distance is 1.84197 (nm) at time 6060 (ps), between atoms 564 and 5318.

What happens if the minimal distance becomes shorter than the cut-off distance used for electrostatic interactions? Is it the case in your simulations?

If the distance would become shorter than the cut-off distance used for electrostatic interactions our energy would dramatically increase. This did not happen for our simulation so we can assume that our measured value of 1.9083 nm is still higher than the cutoff.

Run now g_mindist on the C-alpha group, does it change the results? What does is mean for your system?

With C-alpha group we got a shortest periodic distance is 2.50235 (nm) at time 5690 (ps), between atoms 569 and 5297. This is an increase of the distance since we only consider C-alpha atoms and no side chains as in our previous calculation.

ROOT MEAN SQUARE FLUCTUATIONS

In this part of the analysis we are going to have a look at the root mean square fluctuations. By analyzing this value for our structure we might be able to figure out which parts of our protein are more flexible than others.

 Figure XX: RMSF per residue in R408W Figure XX: RMSF of the backbone per residue in R408W Figure XX: B-factor in R408W
Questions

Indicate the start and end residue for the most flexible regions and the maximum amplitudes.

 Figure XX: Gyration in R408W
Questions

At what time and value does the radius of gyration converge?

STRUCTURAL ANALYSIS: PROPERTIES DERIVED FROM CONFIGURATIONS

SOLVENT ACCESSIBLE SURFACE AREA

 Figure XX: solvent accessible area over time in R408W Figure XX: solvent accessible area per atom in R408W Figure XX: solvent accessible area per residue in R408W

HYDROGEN BONDS

In this part of our analysis we are looking at inter H-bonds within our protein and at H-bonds from our protein to the surrounding solvent. To do so we executed the following command twice:

 Figure XX: intra protein hydrogen bonds in R408W Figure XX: hydrogen bonds between solvent and protein in R408W

SALT BRIDGES

Still running on the lrz.

RMSD matrix

 Figure XX: rmsd matrix of R408W Figure XX: rmsd distribution of R408W

Cluster analysis

 Figure XX: clusters of R408W Figure XX: cluster ids over time of R408W Figure XX: cluster sizes of R408W Figure XX: cluster transitions of R408W

Distance RMSD

 Figure XX: distance RMSD of R408W