Workflow structure based mutation analysis GBA

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Return to structure based mutation analysis of GBA.


SCWRL is a tool, specifically designed for side-chain conformation predictions, given a fixed backbone from an experimental determined structure. <ref>G. G. Krivov, M. V. Shapovalov, and R. L. Dunbrack, Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins (2009). </ref>


  • Create pdb-File only containing chain A of the protein (e.g with pymol): 2NT0_A.pdb
  • Extract sequence (e.g with repairPDB) and change all letters of protein sequence to lower case and introduce mutation in capital letters: mutantSequence.seq
    • Note, that only one mutation should be inserted per sequence -> results in 10 mutant sequence files.
  • Commandline: scwrl4 -i 2NT0_A.pdb -s mutantSequence.seq -o 2NT0_mutant.pdb > scwrl.log


FoldX is an algorithm which provides an estimation of the importance of the interactions contributing to the stability of proteins. It was developped by the FoldX consortium now centered at the Centre de Regulació Genòmica in Barcelona. <ref></ref>


  • Create a runfile according to the example "Energy of the molecule" at the FoldX website: run.txt
    • Do not forget to adjust known information like pH and temperature.
  • Create a list file containing all pdb files you want the energy to be calculated of: list.txt
  • Commandline: sudo ./foldx -runfile run.txt > foldx.output


Minimise is a tool, which attempts to identify the set of rotamers that minimize the model's overall energy.


  • Remove hydrogens and waters from the pdb files with repairPDB: repairPDB mutant.pdb -nosol > mutant_nw.pdb
  • Apply Minimise: minimise mutant_nw.pdb mutant_nw_min.pdb > mutant_nw_min.out


Gromacs is a package which simulates the Newtonian equations of motion, so called molecular dynamics, for large systems and was was first developed in Herman Berendsens group, department of Biophysical Chemistry of Groningen University. <ref></ref>


  • Step 1 - pdb2gmx
    • pdb2gmx adds hydrogens to the molecules and generates a coordinate (.gro) and a topology(.top) file.
    • Commandline: pdb2gmx -f mutant_nw.pdb -o mutant_nw.gro -p -water tip3p -ff amber03
  • Step 2 - MDP file
    • Create a MDP file with the following content:
title = PBSA minimization in vacuum
cpp = /usr/bin/cpp
# Preprocessing
# -DFLEXIBLE: include flexible water in stead of rigid water into topology
# -DPOSRES: includes posre.itp into topology, used for position restraints.
# Implicit solvent - simulation with implicit solvent using the Generalized Born formalism
implicit_solvent = GBSA
# Run control - steepest descent algorithm for energy minimization
integrator = steep
# Run control - tolerance
emtol = 1.0
# Run control - maximum number of steps to integrate or minimize
nsteps = 500
# Output - frequency to write energies to energy file
nstenergy = 1
# Tables - group(s) to write to energy file 
energygrps = System
# Neighbour searching - makes grid in the box and only check atoms in neighboring grid cells when constructing a new neighbor list every nstlist steps
ns_type = grid
# Electrostatics - twin range cut-off's with neighborlist cut-off rlist and Coulomb cut-off rcoulomb
coulombtype = cut-off
rcoulomb = 1.0
# VDW - distance for the LJ or Buckingham cut-off
rvdw	 = 1.0
# Bonds - no constraints except for those defined explicitly in the topology
constraints = none
# Neighbour searching - Use no periodic boundary conditions, ignore the box.
pbc = no
  • Step 3 - grompp
    • grompp, the GROMACS preprocessor, prepares the system for gromacs. Therefore it reads a molecular topology file, checks the validity of the file and expands the topology from a molecular description to an atomic description.
    • Commandline: grompp -v -f mutant_nw.mdp -c mutant_nw.gro -p -o mutant_nw.tpr
  • Step 4 - mdrun
    • The mdrun program is the main computational chemistry engine within GROMACS and can perform molecular dynamics simulations, Brownian Dynamics, Langevin Dynamics, Conjugate Gradient, L-BFGS, or Steepest Descents energy minimization.
    • Commandline: mdrun -v -deffnm mutant_nw
  • Step 5 - g_energy
    • g_energy extracts energy components or distance restraint data from an energy file and calculates Average, RMSD and drift from the simulation.
    • Commandline: g_energy -f mutant_nw.edr -o mutant_nw.xvg
  • Step 6 - trjconv
    • trjconv is able to convert trajectory files in many ways and can be used to create the according pdb files.
    • Commandline: trjconv -f mutant_nw.gro -s mutant_nw.gro -o mutant_nw_gro.pdb.