Canavan Task 8 - Molecular Dynamics Simulations
From Bioinformatikpedia
Protocol
Further information can be found in the protocol.
Choosing Mutants
We decided to use A305E and K213E for the MD analysis. In <xr id="CD_MD_mutants"/>.
<figtable id="CD_MD_mutants">
| Mutation | Comment | Visualisation |
| A305E | A305 is located at the end of the 13th beta sheet at the C-terminus of the protein. In figure <xr nolink id="a305e_crowded"/> the mutated residue glutamic acid is shown in red. The space at this position is rather crowded, so that alanine as a small residue fits very well in this position. Glutamic acid instead, hardly finds space and overlaps with neighboring residues. We decided to use this mutation, since we expect to see a huge effect in structure with the MD simulation. |
<figure id="a305e_crowded"></figure> |
| K213E | K213 is located on the loop connecting the N-, and C-terminal of the enzyme. It is on the surface of the protein, far away from the binding site or the dimer interaction site. In <xr nolink id="k213e_pymol"/>, the mutated residue K213E as computed by PyMol is presented in blue and the reference structure and residue in green. The mutated residue Glutamic Acid is able to form an HBond with the neighbouring helix. We chose this mutation since we do not expect to see any effect from this mutation. Also, our analysis so far suggests, that this mutation is not disease causing. Yet, it is annotated in the HGMD to cause Canavan Disease. We are curious about the MD outcome to either see the HGMD annotation confirmed or our predictions confirmed. | <figure id="k213e_pymol"></figure> |
</figtable>
Running Jobs
We had no problems to get the jobs started.
squeue -u di34fog --cluster mpp1
We started five jobs:
- wt MD run with original 2O4H:A => Run time 05:22:45
- K213E mutant based on Scwrl_min structure => Run time 05:19:05
- K213E mutant based on Foldx_min structure => Run time 05:13:28
- A305E mutant based on Scwrl_min structure => Run time 05:18:20
- A305E mutant based on Foldx_min structure => Run time 05:13:28
Intermediate steps
These are the steps performed by AGros
1
Runs RepairPDB and findBreaks, additional scripts to adapt PDB files to MD reality.
- remove clashing water
- Script: clshwtr.pl
- Out 2O4H_chainA.nclw
- repair pdb
- Script: repairpdb.pl -nohoh
- Out: 2O4H_chainA.pdb
- repair pdb
- Script: repairpdb.pl -jprot
- Out: 2O4H_chainA_br.pdb
- find breaks in structure
- Script: findBreaks.pl
- Out: 2O4H_chainA_br_0.pdb
- repairPDB
- Skript: repairpdb.pl -nodna
- Out: 2O4H_chainA_repair.pdb
2- Runs STRWater, an additional script that saves information for Structural Water Molecules in PDB file. - repairPDB -ssw => _water.pdb, B-Factor is 15 - repairPDB -nodna => _repair.pdb
3- Runs SCWRL, a program employed to correctly assign side chain information to PDB structures. - _nh - _sc => scwrl
4- Runs minimization in vacuum. -pdb2gmx: K213E_scwrl_mini_nh.pdb =>K213E_scwrl_mini.gro =>K213E_scwrl_mini.top
5- Creates water box, includes solvent (water with 0.1 [NaCl]) and neutralizes protein intrinsic charge.
- editconf creates _pbc.gro - genbox creates _water.gro
- grompp -f minim.mdp -c _water.gro -p .top -o _water.tpr - repair_pdb => _solv.pdb
6- Creates restrain files for each chain individually.
- repairPDB -jprot -dna => _solv.pdb2
7- Runs solvent minimization with fixed Protein (backbone + sidechains). _solv_min.tpr Run min I: solvent & fixed protein(SD): => _solv_min.pdb
8- Runs minimization with fixed Backbone. Run min II: solvent & fixed backbone(SD): => _solv_min2.pdb Run min III: solvent & fixed backbone: => _solv_min3.pdb
9- Runs short production NVT MD.
grompp-f nvt.mdp -c _solv_min3.pdb -p _solv.top -o _nvt.tpr _nvt.gro
10- Runs short production NPT MD.
grompp -f /npt.mdp -c _nvt.gro -p _solv.top -o _npt.tpr _npt.gro
11- Runs Production MD. grompp -f md.mdp -c _npt.gro -p _solv.top -o _md.tpr _md.gro
Simulation structure created! ------------------------------------------------------------------------------ Water and box added! ------------------------------------------------------------------------------ Ions added! ------------------------------------------------------------------------------ Number of chains 1 ------------------------------------------------------------------------------ Number of atoms 45678 ------------------------------------------------------------------------------ CHAIN 0: Protein Constraint files created ------------------------------------------------------------------------------ Run min I: solvent & fixed protein(SD) Solvent minimized! ------------------------------------------------------------------------------ Run min II: solvent & fixed backbone(SD) System minimized! ------------------------------------------------------------------------------ Run min III: solvent & fixed backbone(CG) System minimized! ------------------------------------------------------------------------------ Run NVT NVT finished! ------------------------------------------------------------------------------ Run NPT NPT finished! ------------------------------------------------------------------------------ Production run Running: step 2000000, remaining runtime: 0 s Production run finished!
