Difference between revisions of "Structure-based mutation analysis ARSA"

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8. Analyze the minimization of the system with the following command: g_energy -f FILE.edr -o energy_1.xvg. Do the analysis for Bond, Angle and Potential. The xvg graphs can be viewed with xmgrace and in the print settings you can choose eps output, the print and convert to pdf.
 
8. Analyze the minimization of the system with the following command: g_energy -f FILE.edr -o energy_1.xvg. Do the analysis for Bond, Angle and Potential. The xvg graphs can be viewed with xmgrace and in the print settings you can choose eps output, the print and convert to pdf.
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[[Category : Metachromatic_Leukodystrophy]]

Revision as of 15:18, 14 August 2011

Preparation

Visualization with Pymol

The following image shows a pymol visualization of Arylsulfatase A, together with all known active and binding sites.

Pymol visualization of ARSA (with closed gaps). The active site is depicted in yellow, metal-binding site in blue, substrate binding sites in green and missense mutations in red.

One can see, that the mutations are spread out through the protein. Some lie near functional sites, other are very distant from them. The table below shows again Pymol visualizations of all mutations, but each seperately. With this, we want to try to derive investigate the correlation of location of the mutation with respect to functional sites and its effect on the protein function.

Nr. mutation position Pymol image Description Effect on function
1 Asp-Asn 29 Arsa29.png The mutation is located at the position of a metal-binding site. harmful
2 Pro - Ala 136 Arsa136.png The mutation is located near the active site and a substrate binding site in sequence as well as in structure. harmful
3 Gln-His 153 Arsa153.png The mutation is located near the active site and a substrate binding site in sequence as well as in structure. harmful
4 Trp-Cys 193 Arsa193.png The mutation is at moderate distance to all important functional sites of the protein. neutral
5 Thr-Met 274 Arsa274.png The mutation is located very distant from the active site and a substrate binding site in sequence as well as in structure.
It is located within a beta sheet.
harmful
6 Phe -Val 356 Arsa356.png The mutation is at moderate distance to all important functional sites of the protein. neutral
7 Thr-Ile 409 Arsa409.png The mutation is not close to important functional sites. harmful
8 Asn-Ser 440 Arsa440.png The mutation is very distant from all functional sites. neutral
9 Cys-Gly 489 Arsa489.png The mutation is very distant from all functional sites. harmful
10 Arg-His 496 Arsa496.png The mutation is very distant from all functional sites. harmful

Above visualizations indicate, that mutations near functional important sites of the protein are likely to cause a harmful effect. However, for distant mutations no trend can be observed.

SCRWL

First, we extracted the amino acid sequence from our pdb file and converted it to lower case.


repairPDB arsa_model.pdb -seq > arsa.model.seq
tr '[:upper:]' '[:lower:]' < arsa.model.seq > arsa.model.lower.seq

Next, we included the individual mutations as capital letters in seperate files and executed scwrl with the following command:


scwrl cmd

We also ran SCWRL on the wild type (wt) structure in order to make it comparable to energy predictions by other programs. The minimal energy of the graph for the wt is 415.134.

Nr. mutation position Reference amino acid mutated amino acid both (without H-bonds) Minimal energy Energy(mutant)/Energy(wt)
1 Asp-Asn 29 Arsa hydro29.png Arsa scwrl hydro29.png Arsa both29.png 419.996 1.011712
2 Pro - Ala 136 Arsa hydro136.png Arsa scwrl hydro136.png Arsa both136.png 415.133 0.9999976
3 Gln-His 153 Arsa hydro153.png Arsa scwrl hydro153.png Arsa both153.png 420.494 1.012911
4 Trp-Cys 193 Arsa hydro193.png Arsa scwrl hydro193.png Arsa both193.png 416.252 1.002693
5 Thr-Met 274 Arsa hydro274.png Arsa scwrl hydro274.png Arsa both274.png 434.014 1.045479
6 Phe -Val 356 Arsa hydro356.png Arsa scwrl hydro356.png Arsa both356.png 415.134 1
7 Thr-Ile 409 Arsa hydro409.png Arsa scwrl hydro409.png Arsa both409.png 414.481 0.998427
8 Asn-Ser 440 Arsa hydro440.png Arsa scwrl hydro440.png Arsa both440.png 418.863 1.008983
9 Cys-Gly 489 Arsa hydro489.png Arsa scwrl hydro489.png Arsa both489.png 415.136 1.000005
10 Arg-His 496 Arsa hydro496.png Arsa scwrl hydro496.png Arsa both496.png 421.011 1.014157
FoldX

wt: 510.88

Nr. mutation position Minimal energy Energy(mutant)/Energy(wt)
1 Asp-Asn 29 496.88 0.9725963
2 Pro - Ala 136 493.81 0.966587
3 Gln-His 153 493.90 0.9667632
4 Trp-Cys 193 496.23 0.971324
5 Thr-Met 274 503.34 0.9852412
6 Phe -Val 356 495.39 0.9696798
7 Thr-Ile 409 495.45 0.9697972
8 Asn-Ser 440 496.79 0.9724201
9 Cys-Gly 489 495.87 0.9706193
10 Arg-His 496 498.75 0.9762567

Minimise

wt: -3839.492677

Nr. mutation position Free energy Energy(mutant)/Energy(wt)
1 Asp-Asn 29 -4174.487222 1.087250
2 Pro - Ala 136 -4164.523707 1.084655
3 Gln-His 153 -4109.12924 1.070227
4 Trp-Cys 193 -4169.617285 1.085981
5 Thr-Met 274 -4065.730562 1.058924
6 Phe -Val 356 -4109.021939 1.070199
7 Thr-Ile 409 -4121.130656 1.073353
8 Asn-Ser 440 -4120.275589 1.073130
9 Cys-Gly 489 -4127.969116 1.075134
10 Arg-His 496 -4120.151911 1.073098


Gromacs

Gromacs is an abbreviation for 'GROningen Mixture of Alchemy and Childrens' Stories' and is a package to perform molecular dynamics. It is Free Software, available under the GNU General Public License.

Workflow

1. Use fetchpdb to get the pdb structure – look at the script, what does it do?

-> we did not use fetchpdb, as we already had the needed pdb-file. The script simply downloads a pdb-file for a give PDB-ID from the PDB-webpage.


2. Use repairPDB to clean the PDB and extract the protein only - describe what options you chose and what other options are available. Make sure you chose the right chain.

-> TODO BENE


3. Run SCWRL with the lowercase protein sequence to make sure there are no missing sidechains. The sequence can be extracted using repairPDB. After SCWRL you will have to remove the hydrogens again.

-> TODO BENE


4. Use the gromacs command “pdb2gmx” the option –f defines the input structure, -o the gromacs outputfile (.gro) and –p the topology (.top) output file. IMPORTANT the input must end with “pdb”. Choose a forcefield and for water the TIP3P model.

-> we chose the AMBER03 forcefield as it is optimized for the use with proteins


5. Create a MDP file with the following content

title = PBSA minimization in vacuum
cpp = /usr/bin/cpp
define = -DFLEXIBLE -DPOSRES
implicit_solvent = GBSA
integrator = steep
emtol = 1.0
nsteps = 500
nstenergy = 1
energygrps = System
ns_type = grid
coulombtype = cut-off
rcoulomb = 1.0
rvdw	 = 1.0
constraints = none
pbc = no

Give a brief description of the different keywords used in this file - for this you should use the gromacs manual.

-> DFLEXIBLE: water in the the topology is flexible
-> DPOSRES: position restraints, defined in posre.itp, are included in the topology
-> implicit_solvent = GBSA: defines the implicit solvent model, here the Generalized Born formalism is used
-> integrator = steep: defines the algorithm for energy minimization, here the steepest descent algorithm is used
-> emtol: defines, when convergence is assumed: minimization is stoppen when maximum force is smaller than this value
-> nsteps: defines the maximum number of steps in the energy minimization
-> nstenergy: frequency in which energies are written to the energy file
-> energygrps: group(s) to write to the energy file 
-> ns_type: defines type of Neighbor searching, here a grid is built and only atoms in neighboring grid cells are checked when a new neighbor list is constructed
-> coulombtype: Twin range cut-off’s with neighborlist cut-off rlist and Coulomb cut-off rcoulomb, where rcoulomb ≥ rlist.
-> rcoulomb: distance for the Coulomb cut-off
-> rvdw: distance for the LJ or Buckingham cut-off
-> constraints: defines additional constraints besides the ones defined in the topology file
-> pbc: Use no periodic boundary conditions, ignore the box.


6. Use grompp to prepare the system for gromacs: grompp -v -f FILE.mdp -c FILE.gro -p FILE.top -o FILE.tpr FILE.tpr is the system file to create which we use in the next step

-> commandline: grompp -v -f gromacs_AMBER03.mdp -c gromacs_AMBER03.gro -p gromacs_AMBER03.top -o gromacs_AMBER03.tpr


7. Now we minimize the system: mdrun -v -deffnm FILE

-> commandline: mdrun -f -deffnm gromacs_AMBER03


8. Analyze the minimization of the system with the following command: g_energy -f FILE.edr -o energy_1.xvg. Do the analysis for Bond, Angle and Potential. The xvg graphs can be viewed with xmgrace and in the print settings you can choose eps output, the print and convert to pdf.