Molecular Dynamics Analysis BCKDHA
Contents
- 1 A brief check of results
- 1.1 How many frames are in the trajectory file and what is the time resolution?
- 1.2 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?
- 1.3 Which contribution to the potential energy accounts for most of the calculations?
- 2 Visualization of results
- 3 Quality assurance
- 4 Structural analysis
- 5 Analysis of dynamics and time-averaged properties
A brief check of results
To verified that the simulations finished properly we first use the command
gmxcheck -f wt.xtc
How many frames are in the trajectory file and what is the time resolution?
- frames: 2001
- time resolution: 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?
- real time: 9h27:35
- simulation speed: 25.370 ns/day
- simulation speed: 107991 years/second
Which contribution to the potential energy accounts for most of the calculations?
- potential energy: -1.24431e+06
Visualization of results
To get a pdb file to be able to visualize the model with pymol we used the Swiss army knife gromacs tool trjconv:
trjconv -s wt.tpr -f wt.xtc -o protein.pdb -pbc nojump -dt 10
Quality assurance
Energy calculations
To calculate the different energies we used the command:
g_energy -f wtMD.edr -o energy.xvg
After submitting this command we had to choose the energy which should calculated.
- Pressure: 13
- Temperature: 12
- Potential: 9
- Total Energy: 11
Pressure
|
In Figure 2 the pressure of the molecular dynamic system is shown. The average value is 1.0161 bar which is shown in the table above. But as we can see the pressure ranges from about -250 bar to 250 bar. Since there is such a big range of 500 bar we are not sure if this value of 1.0161 bar is the equilibrium which should be reached or only the arithmetic average of all the values.
Temperature
|
In Figure 3 the temperature of the MD simulation is shown. As we can see it ranges between 294 K and 302 K so it has a very small deviation of the average value of 297.9 K. Since there is only such a small fluctuation we can see that the temperature in the system is quite stable which means that it reached an equilibrium.
Potential
|
Figure 4 shows the potential of the md system. As we can see in the picture the potential ranges from -1.24e+06 kJ/mol to -1.25e+06 kJ/mol. Although this is a very huge range of 10000 we can see that all in all the potential is very low. This low potential indicates that the protein is quite stable. Since the structure of a protein is responsible for the functino of a protein this stability is important for the function of the protein.
Total Energy
|
The low potential energy already indicated that the total energy of the system has to be quite low. By looking at Figure 5 we can see that the energy is a bit higher than the potential energy but it is still very low. Additionally there is less variation in the energy since it ranges between -1.017e+06 kJ/mol and -1.025e+06 kJ/mol. Again we can say that such a low energy stands for a stable protein which indicates that the simulation was correct.
Minimum distance between periodic boundary cells
To calculate the minimum distance we used the command
g_mindist -f wtMD.xtc -s wtMD.tpr -od minimal-periodic-distance.xvg -pi
After submitting this command we chose group 1 to calculate the minimum distance for the whole protein.
The shortest periodic distance is 1.40945 (nm) at time 6090 (ps) between atoms 25 and 6490.
Root mean square fluctuations
For calculating the RMSF of a protein each atom of this protein is compared with the calculated average stucture of the protein. By comparing them it is possible to find out how much it varies from its average position and so the flexibility of this region can be calculated. Regions with a high fluctuation are more flexible than regions with a low one.
To calculate the minimum distance we used the command
g_rmsf -f wtMD.xtc -s wtMD.tpr -o rmsf-per-residue.xvg -ox average.pdb -oq bfactors.pdb -res
After submitting this command we had to choose the group we want the RMSF to be calculated for:
- Protein: 1
- C-alpha: 3
RMSF for protein | RMSF for C-alpha |
---|---|
Figure 7 where the RMSF for the whole protein is plotted points out that mainly the beginning of the protein features the most fluctuation. This indicates that this region of the protein is the one with the most flexibility. In the middle part of the protein there are nearly no peaks which means that this part is fixed. In the end there is a peak which is a bit higher than the rest of the protein. This could suggest that this region is also a bit flexible.
In Figure 8 we did the RMSF calculations not for the whole protein but only for the c-alpha atoms. Those atoms are the central carbon atoms of the protein which means that in this case calculation only the backbone is considered. But by comparing the two plots above with each other we can see that in both cases the beginning of the protein is the part which has definitly the huges fluctuation of the average structure. So not only the side chains differ from the average structure but also the backbone which indicates for a strong flexibility.
Pymol analysis of average and bfactors
These average.pdb file was produced automatically during the calculation of the RMSF because it is needed for comparisons. This file contains the average structure of the protein. Because of the option -oq the bfactor.pdb file was produced additionally. In this file the temperature factors (bfactors) are calculated and added to a reference structure. Normally the parts of the protein which are most flexible have also the highest temperature. To find out if this is the fact in our case we used pymol to analyze the average structure and the bfactors structure. Additionally we compared the predicted average structure with the original structure of our protein.
Protein
1u5b/average | 1u5b/bfactors | bfactors/average |
---|---|---|
RMSD: 1.169 | RMSD: 0.377 | RMSD: 1.422 |
To find out how accurate the calculated average structure is we aligned it with the original structure of out protein (1u5b). As we can see in Figure 9 and additionally because of the RMSD value of 1.169 the superposition of the two structures is not covering perfectly. The middle part of the protein is aligned quite good. The most deviating parts are the two ends of the structures on the left and on the right side of the picture.
C-alpha
1u5b/average | 1u5b/bfactors | bfactors/average |
---|---|---|
RMSD: 0.955 | RMSD: 0.300 | RMSD: 0.993 |
Radius of gyration
To calculate the radius of gyration we used the command
g_gyrate -f wtMD.xtc -s wtMD.tpr -o radius-of-gyration.xvg
After submitting this command we chose group 1 to calculate the radius of gyration for the whole protein
Structural analysis
First we had to use the command
trjconv -f wtMD.xtc -o wtMD_nojump.xtc -pbc nojump
This is important because the protein possibly jumpes out of the box so the trajectory has to be rebuild. This has the effect that the particles are back in the center.
Solvent accessible surface area
To calculate the solvent accessible surface area we used the command
g_sas -f wtMD_nojump.xtc -s wtMD.tpr -o solvent-accessible-surface.xvg -oa atomic-sas.xvg -or residue-sas.xvg
After submitting this command we had to choose two groups. Both times we chose protein.
Solvent accessible surface | SAS over time per residue | SAS over time per atom |
---|---|---|
Hydrogen bonds
To calculate the hydrogen bonds between protein and protein and between protein and water we used the commands
echo 1 1 | g_hbond -f wtMD_nojump.xtc -s wtMD.tpr -num hydrogen-bonds-intra-protein.xvg
echo 1 12 | g_hbond -f wtMD_nojump.xtc -s wtMD.tpr -num hydrogen-bonds-protein-water.xvg
protein and protein | protein and water |
---|---|
salt bridges
Ramachandran plot
To calculate the ramachandran plot we used the command
g_rama -f wtMD_nojump.xtc -s wtMD.tpr -o ramachandran.xvg
Analysis of dynamics and time-averaged properties
RMSD matrix
To calculate the RMSD matrix we used the command
g_rms -s wtMD.tpr -f wtMD_nojump.xtc -f2 wtMD_nojump.xtc -m rmsd-matrix.xpm -dt 10
After submitting this command we had to choose two groups. Both times we chose protein.
- Minimum: 0.000
- Maximum: 0.579
- Average: 0.329
cluster analysis
To calculate the cluster we used the command
echo 6 6 | g_cluster -s wtMD.tpr -f wtMD_nojump.xtc -dm rmsd-matrix.xpm -dist rmsd-distribution.xvg -o clusters.xpm -sz cluster-sizes.xvg -tr cluster-transitions.xpm -ntr cluster-transitions.xvg -clid cluster-id-over-time.xvg -cl clusters.pdb -cutoff 0.1 -method gromos -dt 10
For the analysis of the first two cluster we used pymol.
542 cluster
RMSD cluster1+2: 0.709
internal RMSD
To calculate the internal RMSD we used the command
g_rmsdist -s wtMD.tpr -f wtMD_nojump.xtc -o distance-rmsd.xvg
After submitting this command we chose group 1 to calculate the internal RMSD for the protein
- rmsmax: -1000
- rmscmax: inf