Canavan Task 8 - Molecular Dynamics Simulations

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">

<xr nolink id="CD_MD_mutants"/> Description of the structural environment of each mutation and visualization of the pymol mutation and the scwrl mutation output.

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"> <xr nolink 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"> <xr nolink 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 and 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
  • Skript: repairpdb.pl -ssw (The default cutoff value for B-Factor is 15)
  • Out: 2O4H_chainA_water.pdb

• repairPDB
  • Skript: repairpdb.pl -nodna
  • Out: 2O4H_chainA_dna.pdb

3

Runs SCWRL, a program employed to correctly assign side chain information to PDB structures.

• Create Seq File for Scwrl
  • Out: 2O4H_chainA.seq

• Run Scwrl
  • Script: scwrl -i _repair.pdb -o _sc.pdb -s .seq
  • Out: 2O4H_chainA_sc.pdb

• Get rid of hydrogen atoms
  • Script: repairpdb.pl -jprot
  • Out: 2O4H_chainA_nh.pdb

4

Runs minimization in vacuum.

• pdb2gmx
  • Script: pdb2gmx -f _nh.pdb -o .gro -p .top -water tip3p -ff amber03 -vsite hydrogens -chainsep id_or_ter
  • Out: 2O4H_chainA.gro, 2O4H_chainA.top

5

Creates water box, includes solvent (water with 0.1 [NaCl]) and neutralizes protein intrinsic charge.

• Define solvent box with 1 nm (10Å) distance to border and add solvent. A dodecahedric box is defined by default
  • editconf -f .gro -o _pbc.gro -bt $boxtype -d 1.0
  • 2O4H_chainA_pbc.gro

• • genbox -cp _pbc.gro -cs spc216.gro -p .top -o _water.gro
  • Out: 2O4H_chainA_water.gro

• Fill it with water, neutralize the system and add ions to 0.1 NaCl concentration
  • minim.mdp
  • grompp -f minim.mdp -c _water.gro -p .top -o _water.tpr
  • Out: 2O4H_chainA_water.tpr

• Add ions
  • genion -s _water.tpr -o _solv_tmp.pdb -conc 0.1 -neutral -pname NA+ -nname CL
  • Out: 2O4H_chainA_solv.pdb
  • Out: 2O4H_chainA_solv.top

6

Creates restrain files for each chain individually.

• genrestr -f _solv.pdb -o .itp
  • Out: posre.itp

7

<figure id="solv_min">

<xr nolink id="solv_min"/>
Step 7: _solv_min.pdb

</figure>

Runs solvent minimization with fixed Protein (backbone + sidechains).

• 2O4H_chainA_solv_min.tpr
  • minim_solv.mdp
  • grompp -f minim_solv.mdp -c _solv.pdb -p _solv.top -o _solv_min.tpr
  • Out: 2O4H_chainA_solv_min.tpr
  • Out: 2O4H_chainA_solv_min.pdb

8

<figure id="solv_min2">

<xr nolink id="solv_min2"/>
Step 7: _solv_min2.pdb

</figure>

Runs minimization with fixed Backbone.

• Run min II: solvent & fixed backbone(SD)
  • grompp -f minim_solv.mdp -c _solv_min.pdb -p _solv.top -o _solv_min2.tpr
  • Out: 2O4H_chainA_solv_min2.tpr
  • Out: 2O4H_chainA_solv_min2.pdb

• Run min III: solvent & fixed backbone
  • grompp -f minim_solv.mdp -c __min2.pdb -p _solv.top -o _solv_min3.tpr
  • Out: 2O4H_chainA_solv_min3.tpr
  • Out: 2O4H_chainA_solv_min3.pdb

<figure id="solv_min3">

<xr nolink id="solv_min3"/>
Step 7: _solv_min3.pdb

</figure>

9

Runs short production NVT MD.

• nvt.mdp
• grompp-f nvt.mdp -c _solv_min3.pdb -p _solv.top -o _nvt.tpr
  • Out: 2O4H_chainA_nvt.tpr
  • Out: 2O4H_chainA_nvt.gro

10

Runs short production NPT MD.

• npt.mdp
• grompp -f npt.mdp -c _nvt.gro -p _solv.top -o _npt.tpr
  • Out: 2O4H_chainA_npt.tpr
  • Out: 2O4H_chainA_npt.gro

11

Runs Production MD.

• Finally MD simulation
• md.mdp
• grompp -f md.mdp -c _npt.gro -p _solv.top -o _md.tpr
  • Out: 2O4H_chainA_md.tpr
  • Out: 2O4H_chainA_md.grole