Canavan Task 8 - Molecular Dynamics Simulations

From Bioinformatikpedia


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"/>
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"/>


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


Runs RepairPDB and findBreaks and additional scripts to adapt PDB files to MD reality.

  • remove clashing water
    • Script:
    • Out 2O4H_chainA.nclw
  • repair pdb
    • Script: -nohoh
    • Out: 2O4H_chainA.pdb
  • repair pdb
    • Script: -jprot
    • Out: 2O4H_chainA_br.pdb
  • find breaks in structure
    • Script:
    • Out: 2O4H_chainA_br_0.pdb
  • repairPDB
    • Skript: -nodna
    • Out: 2O4H_chainA_repair.pdb


Runs STRWater, an additional script that saves information for Structural Water Molecules in PDB file.

  • repairPDB
    • Skript: -ssw (The default cutoff value for B-Factor is 15)
    • Out: 2O4H_chainA_water.pdb
  • repairPDB
    • Skript: -nodna
    • Out: 2O4H_chainA_dna.pdb


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: -jprot
    • Out: 2O4H_chainA_nh.pdb


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,


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
    • Setup the Box for your simulations
    • Skript: editconf -f .gro -o _pbc.gro -bt $boxtype -d 1.0
    • Output: 2O4H_chainA_pbc.gro
    • Solvate the Box
    • Skript: 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:


Creates restrain files for each chain individually.

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


Runs solvent minimization with fixed Protein (backbone + sidechains). See <xr id="solv_min"/>.

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

<figure id="solv_min">

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



Runs minimization with fixed Backbone. See <xr id="solv_min2"/>.

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

<figure id="solv_min2">

<xr nolink id="solv_min2"/>
Step 8: _solv_min2.pdb


  • Run min III: solvent & fixed backbone. See <xr id="solv_min3"/>.
    • grompp -f minim_solv.mdp -c __min2.pdb -p -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 9: _solv_min3.pdb



Runs short production NVT MD.

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


Runs short production NPT MD.

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


Runs Production MD.

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

Trajectory visualisation

We were too much in love with the Fabry Disease Animation, so we shamelessly followed their instructions in the journal to see how it works and have one of our own. Sorry and thanks for putting them online!

Animation in <xr id = "k213e"/>.

Note how our protein is not in the center of the simulation box, but at the spheres, which makes you see the periodic boundary conditions used for the MD simulation.

<figure id="k213e">

<xr nolink id="k213e"/>
Trajectory visualisation