Protocol BCKDHA MD

A brief check of results

To verified that the simulations finished properly we first use the command

• gmxcheck -f wt.xtc

Visualization of results

We used the Swiss army knife gromacs tool trjconv to get a pdb file which we could use to visualize the model with pymol :

• trjconv -s wt.tpr -f wt.xtc -o protein.pdb -pbc nojump -dt 10

To visualize the model we loaded it with pymol and used the following commands:

• mplay
• spectrum
• show cell
• intra_fit protein
• orient
• dss

Since pymol was killed everytime we wanted to go on we saved the movie as png images at this step.
To create a gif we wrote a script. In this script we first converted the filetype from .png in .jpg. For the movie we only used every thirtythird image so that the movie was small enough. To create a movie out of these images we used the command:

• convert -delay 30 -loop 0 *.jpg animation.gif

Quality assurance

Energy calculations

To calculate the different energies we used the following 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

Minimum distance between periodic boundary cells

The determination of the minimum distance between periodic boundary cells is a crucial part in the quality assurance of an MD simulation. In this step you have to verify that there were no direct interactions between periodic images, as interactions between atoms of the same molecule over the periodic boundary would disturb the native behaviour of the protein. This would lead to invalid molecular dynamics results. Therefore we have to check that the minimum distance is greater than 0.9.

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.

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

Radius of gyration

The radius of gyration reflects how the structure changes during the simulation and how the shape changes during the time.

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 jumps 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

The solvent accessible surface area (SASA) of a protein is the part of the surface which is reachable a solvent. This definition of SASA can be devided into two subgroups - hydrophilic SASA and hydrophobic SASA. This shows that the possibility that a solvent can reach the surface depends on its properties.

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.

Hydrogen bonds

There are two different possibilities of hydrogen bonds. They can be inside of the protein (protein-protein) or between the protein and the surrounding solvents. For the building of a hydrogen bond it is important that the hydrogen-donor and the hydrogen-acceptor are not to far away from each other. This means that high flexibility of a protein would lead to high variation in the 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

Ramachandran plot

In a Ramachandran plot the backbone dihedral angles ψ and φ of the amino acid residues are visualized. On the left upper corner the beta sheets are shown. Under the beta sheets the alpha helices are described. On the right side the lefthanded helices of the protein are shown.

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

A RMSD matrix is helpful to find groups of structures which are similar between the different points of time. When there are groups of structures which are similar the RMSD value is lower between them and high to other groups. The range of the RMSD value is from 0 (blue) to 0.579 (red).

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.

Cluster analysis

The similarity of structures which is analysed above can also be calculated and shown by clustering the structures which are similar to each other. This is done in the next step.

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.

Internal RMSD

The internal RMSD are the atomic distances inside the protein. With this measure it is possible to get information about the changes of the structure during the simulation.

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

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