Structure-based mutation analysis BCKDHA

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Revision as of 14:01, 30 June 2011 by Demel (talk | contribs) (Y166N)

Structure selection

The following table presents the PDB structures for BCKDHA to date:

PDB id resolution [Å] R-factor coverage ph-value
1DTW 2.70 0.224 7.5*
1OLS 1.85 0.172 5.5
1OLU 1.90 0.161 5.5
1OLX 2.25 0.161 5.5
1U5B 1.83 0.156 5.8
1V11 1.95 0.139* 5.5
1V16 1.90 0.132* 5.5
1V1M 2.00 0.130* 5.5
1V1R 1.80 0.158 5.5
1WCI 1.84 0.149 5.5
1X7W 1.73 0.148 5.8
1X7X 2.10 0.149 5.8
1X7Y 1.57 0.150 5.8
1X7Z 1.72 0.154 5.8
1X80 2.00 0.161 5.8
2BEU 1.89 0.171 5.5
2BEV 1.80 0.139 5.5
2BEW 1.79 0.147 5.5
2BFB 1.77 0.145 5.5
2BFC 1.64 0.144 5.5
2BFD 1.39* 0.150 5.5
2BFE 1.69 0.150 5.5
2BFF 1.46 0.150 5.5
2J9F 1.88 0.171 5.5

The following PDB Structure was chosen because of its good experimental resolution: <bold></bold>

  • resultion:
  • R-factor
  • ph-value

Comparison energies

Mapping of the mutations on the crystal structure


Before we could use SCWRL we first had to get the sequence of our model: repairPDB bckdha.pdb -seq >> bckdha.seq

When we have the sequence we have to make one file for each mutation. In these files we copied the bckdha.seq and changed the sequence to lower case letters. Then we add the mutation in an upper case letter.

To run SCWRL we used the command: scwrl -i bckdha.pdb -s mutation1.seq -o mutation1Model.pdb

Total minimal energy of the graph

Position Energy
M82L 642.213
Q125E 616.85
Y166N 616.293
G249S 633.378
C264W 805.257
R265W 710.647
I326T 619.424
F409C 617.305
Y438N 615.951


To use foldX we first build a runscript. Additionally we had to create one file with all PDB Ids each in a new line (list.txt). We used the command Foldx -runfile run.txt > Stout.txt to run the programm.


total energy difference
wildtype 401.00 0
mutant1 437.88 -36.88
mutant2 431.77 -30.77
mutant3 432.24 -31.24
mutant4 432.22 -31.22
mutant5 488.43 -87.43
mutant6 460.43 -59.43
mutant7 432.94 -31.94
mutant8 433.33 -32.33
mutant9 431.56 -30.56

After using foldx we have the total energy for the wiltype protein and for each mutation. The value of the wildtype protein is 401.00 which is already a high value. This means that the protein is quite instabile. To find out which mutation has a high influence on the protein we look at the energies and especially on the difference between the energy of the mutated protein and the wildtype protein. All of the proteins have a higher energy than the unmutated protein which means that these proteins are less stable.


Bevore using Minimise it is important to remove the hydrogens and water has to be removed. For this we used the new version of repairPDB of the virtualbox. The programm can be started with the command: repairPDB bckdha.pdb -nosol out.pdb > Stout.txt
It is useful to save the output in a file because it includes the energy.



The first part describes general background information for gromacs as well as how to run those programs. The second part contains the result description and analysis.


1. fetchpdb

The fetch-pdb script first checks, if it was called with an valid PDB-id. If the entered PDB code has 4letters, the script tries to download the pdb-file from the server. The successfully downloaded folder gets unzipped and everything except the actual pdb file is removed.

2. repairPDB

repairPDB bckdha_mod.pdb -noh -nosol > bckdha_clean.pdb


scwrl -i bckdha_mod.pdb -s extractedPDB.seq -o bckdha_scwrl.pdb

pdb including HEATOMS


use clean pdb without HEATOMS

pdb2gmx -f bckdha_clean.pdb -o bckdha.gro -p -water tip3p -ff amber03

5. MDP

6. grompp

grompp -v -f MDP_bckdha.mdp -c bckdha.gro -p -o bckdha.tpr

7. System Minimization

mdrun -v -deffnm bckdha 2> mdrun_out.txt

8. Analyzation

g_energy -f bckdha.edr -o energy_1.xvg


Wildtype analysis: nsteps vs time

steps time (real) [s]
50 8.074
100 10.362
500 6.156
1000 15.240
5000 4.231

Wildtype analysis: force fields

The different force fields chosen for this task were:

  • AMBER03
  • CHARMM27

Mutation analysis


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2518.71 1700 6337.97 -10023.3
Angle 3642.41 270 638.624 -1479.34
Potential 5.16e+06 5.1e+06 7.47e+07 -3.13e+07


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2519.85 1700 6351.32 -10027.5
Angle 3626.21 260 618.433 -1418.24
Potential 5.23e+06 5.2e+06 7.5e+07 -3.17e+07


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3029.19 2200 -nan -12529.5
Angle 3654.58 280 -nan -1486.71
Potential 7.95e+06 7.8e+06 -nan -4.67e+07


Energy Average Err.Est RMSD Tot-Drift


Energy Average Err.Est RMSD Tot-Drift


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2473.43 1700 6385.14 -9741.04
Angle 3726.4 330 827.187 1803.54
Potential 5.36e+06 5.3e+06 7.68e+07 -3.26e+07


Energy Average Err.Est RMSD Tot-Drift


Energy Average Err.Est RMSD Tot-Drift


Energy Average Err.Est RMSD Tot-Drift


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