Difference between revisions of "Canavan Task 8 - Molecular Dynamics Simulations"

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Runs minimization in vacuum.
 
Runs minimization in vacuum.
-pdb2gmx:
 
K213E_scwrl_mini_nh.pdb
 
=>K213E_scwrl_mini.gro
 
=>K213E_scwrl_mini.top
 
   
   
  +
*pdb2gmx
5- Creates water box, includes solvent (water with 0.1 [NaCl]) and neutralizes protein intrinsic charge.
 
  +
**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
   
- editconf creates _pbc.gro
 
- genbox creates _water.gro
 
   
  +
===5===
- grompp -f minim.mdp -c _water.gro -p .top -o _water.tpr
 
- repair_pdb => _solv.pdb
 
 
 
   
  +
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
6- Creates restrain files for each chain individually.
 
  +
**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
- repairPDB -jprot -dna => _solv.pdb2
 
  +
**Skript: genbox -cp _pbc.gro -cs spc216.gro -p .top -o _water.gro
  +
**Out: 2O4H_chainA_water.gro
   
  +
7- Runs solvent minimization with fixed Protein (backbone + sidechains).
 
  +
*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===
  +
  +
Runs solvent minimization with fixed Protein (backbone + sidechains). See <xr id="solv_min"/>.
 
 
  +
*2O4H_chainA_solv_min.tpr
_solv_min.tpr
 
  +
**minim_solv.mdp
Run min I: solvent & fixed protein(SD): => _solv_min.pdb
 
  +
**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
   
  +
<figure id="solv_min">[[File:K213E_scwrl_mini_solv_min.png|thumb|center|200px|<b><xr nolink id="solv_min"/> <br>Step 7: _solv_min.pdb</b>]]</figure>
   
  +
===8===
8- Runs minimization with fixed Backbone.
 
  +
  +
Runs minimization with fixed Backbone. See <xr id="solv_min2"/>.
 
 
Run min II: solvent & fixed backbone(SD): => _solv_min2.pdb
 
Run min III: solvent & fixed backbone: => _solv_min3.pdb
 
   
  +
*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
   
  +
<figure id="solv_min2">[[File:K213E_scwrl_mini_solv_min2.png|thumb|center|200px|<b><xr nolink id="solv_min2"/> <br>Step 8: _solv_min2.pdb</b>]]</figure>
9- Runs short production NVT MD.
 
   
grompp-f nvt.mdp -c _solv_min3.pdb -p _solv.top -o _nvt.tpr
 
_nvt.gro
 
   
  +
*Run min III: solvent & fixed backbone. See <xr id="solv_min3"/>.
  +
** 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">[[File:K213E_scwrl_mini_solv_min3.png|thumb|center|200px|<b><xr nolink id="solv_min3"/> <br>Step 9: _solv_min3.pdb</b>]]</figure>
10- Runs short production NPT MD.
 
   
  +
===9===
grompp -f /npt.mdp -c _nvt.gro -p _solv.top -o _npt.tpr
 
_npt.gro
 
   
  +
Runs short production NVT MD.
11- Runs Production MD.
 
  +
 
  +
*nvt.mdp
grompp -f md.mdp -c _npt.gro -p _solv.top -o _md.tpr
 
  +
*grompp-f nvt.mdp -c _solv_min3.pdb -p _solv.top -o _nvt.tpr
_md.gro
 
  +
**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
  +
  +
  +
=== Trajectory visualisation ===
  +
  +
We were too much in love with the [https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php/Fabry:Molecular_Dynamics_Simulations#Trajectory 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">[[File:k213e.gif|thumb|left|400px|<b><xr nolink id="k213e"/> <br>Trajectory visualisation</b>]]</figure>
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!
 

Latest revision as of 15:38, 3 July 2012

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
    • 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: 2O4H_chainA_solv.top

6

Creates restrain files for each chain individually.

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

7

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 _solv.top -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

</figure>

8

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 _solv.top -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

</figure>


  • Run min III: solvent & fixed backbone. See <xr id="solv_min3"/>.
    • 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 9: _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


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

</figure>