Molecular Dynamics Simulations TSD
The journal for this task can be found here.
Contents
Mutations
Based on previous analyses the following two mutations were chosen for the MD analysis.
R178H because it is a disease causing mutation with a strong effect on the active site. R178C was not chosen, since there seems to be no possibility to make it work at all, while for the mutation to histidine it should be possible ot fit the residue in the binding pocket. This will still not allow coordination of the ligand, so the mutation is disease-causing for sure, but it will still be interesting to see where the sidechain is placed and what alternative interactions might arise.
The second mutation is P182L. The residue is found at the protein surface, near the binding site to the beta-subunit. However it does not seem to play a role in the interaction. It will be interesting to see how this changes due to MD simulation. In addition, it should be oberserved how the helix changes, now that the helix breaker proline is not present anymore.
Simulations
Intermediate Structures
In the AGroS pipeline several structures are created in intermediate steps. <xr id="tbl:intermed"/> lists all of these and gives an explanation of their content.
<figtable id="tbl:intermed">
File | Description | Scripts |
---|---|---|
2gjx178H | Input structure, with waters removed | repairdPDB.sh -nohoh, clshwtr.pl with cutoff 2.6 |
_br | Protein part only | repairdPDB.sh -jprot |
_br_0 | Structure until the first break. Since there are none, it is the full structure again | findBreaks.pl |
.pdb2 | 2gjx178H.pdb with hydrogens removed | repairPDB -noh |
_repair | Indices changed to begin with 0 and DNA removed | repairPDB -nodna -offset |
_repair_0 | Recalculated indices, if several chains present in input | sepPDB, repairPDB -rres |
_water | Waters with B-value below 15 Å extracted from the structure | repairPDB -ssw 15 |
_dna | DNA extracted from the structure | repairPDB -dna |
_sc | Side chain placements by SCWRLv4 | scwrl |
_nh | Protein part only from _sc | repairPDB -jprot |
_solv_tmp | Addition of ions (Na+ and Cl-) | genion -pname NA+ -nname CL- |
_solv | Remove overlapping solvents from _solv_tmp | repairPDB -cleansol |
_solv.pdb2 | DNA and protein part only from _solv_tmp | repairPDB -dna, repairPDB -jprot |
solv_0 | Input for creation of custom position restraints for several chains, if present | ? |
_solv_min | Minimization run with only solvent flexible. Protein backbone and sidechains are fixed | grompp, mdrun |
_solv_min.pdb2 | Protein only extracted from _solv_min.pdb | repairPDB -jprot |
_solv_min_0 | Input for creation of custom position restraints for several chains, if present | ? |
_solv_min2 | Minimization with only backbone fixed | grompp, mdrun |
_solv_min3 | Another minimization with fixed backbone, this time using conjugate gradient as integrator | grompp, mdrun |
step *? |
</figtable>
The state of the job and whether it really sits in the queue can be checked with the command squeue -u <username> <queue> where the queue can either be --clusters=mpp1 or --partition=mpp1_inter.
Once this all worked you have to wait and write a bit about the different steps of the simulation etc.
We also want you to look at the intermediate PDB files created in the workflow, visualize them and explain what is special, different about them and why we need them.
Final structures
Wildtype
P182L
R178H
References
<references/>