Structure-based mutation analysis BCKDHA

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Revision as of 18:13, 1 July 2011 by Demel (talk | contribs) (Mapping of the mutations on the crystal structure)

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

We could not use any of the PDB structures for BCKDHA because all of them had gaps in the secondary structure which means that some residues were missing. So we took the structure which has the less gaps: 1U5B

  • resultion: 1.83
  • R-factor: 0.156
  • ph-value: 5.8

This structure has to be modified with some programms to close the gaps. Additionally the first residues which are in BCKDHA misses in 1U5B thats why the startposition corresponds to position 6 of the BCKDHA sequence.

As we can see non of the values correspond to the demand because it was ask for an structure which has a very small R-factor, a pH of 7.4 and a high resolution.

Mapping of the mutations on the crystal structure

Hydrogen Bonds for amino acid 82 in the wild type structure
File:BCKDHA Mut37.jpg
Hydrogen Bonds for amino acid 82 in the mutated type structure

Comparison energies


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. It is important to change values of <Temperature> and <pH> to the values of the used protein. These values can be found on the pdb side . 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
M82L 437.88 -36.88
Q125E 431.77 -30.77
Y166N 432.24 -31.24
G249S 432.22 -31.22
C264W 488.43 -87.43
R265W 460.43 -59.43
I326T 432.94 -31.94
F409C 433.33 -32.33
Y438N 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 mutated proteins have a much higher energy than the unmutated protein which means that these proteins are less stable. We can see in the table that the proteins can be divided into two groups. The first group has an energy difference of about 31 and the other group has a much higher difference.


It is important to remove the hydrogens and water before using the programm. 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 also possible to use the old version but then the command is: repairPDB bckdha.pdb -nosol -noh out.pdb > Stout.txt
It is useful to save the output in a file because it includes the energy.

total energy difference
wildtype -2485.452755 0
M82L -4253.174790 1767.722015
Q125E -4080.989512 1595.536757
Y166N -4354.495238 1869.042483
G249S -4280.043000 1794.590245
C264W -3745.313620 1259.860865
R265W -3989.790625 1504.33787
I326T -4317.105618 1831.652863
F409C -4358.528143 1873.075388
Y438N -4339.778964 1854.326209

Minimise calculates the energy for a mutation by building a new model for each mutation. And then it calculates the energy for the whole mutated model. To find out if there is a difference between the wildtype and the model that is calculated by Minimise. The aim by comparing the mutated models with the wildtype is to find out if there is a structural change caused by a mutation. We superposed each mutated protein with the wildtype and focused on the mutated position. In the pictures there are always the superposed structures. In the wildtype pictures the structure of the unmutated residue is bold and in the mutated pictures the structure of the mutated residue is bold. So we can compare the two pictures to see if there is a change in the structure caused by the mutation on this residue.

mutation wildtype structure mutated structure
mutation M82L
mutation Q125E
mutation Y166N
mutation G249S
mutation C264W
mutation R265W
mutation I326T
mutation F409C
mutation Y438N


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
GROMACS Energy for the AMBER03 forcefield using the wildtype bckdha structure.
  • CHARMM27
GROMACS Energy for the CHARMM27 forcefield using the wildtype bckdha structure.
GROMACS Energy for the OPLS-AA forcefield using the wildtype bckdha structure.

Bond Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 3072.83 2200 -nan -13100.2
CHARMM25 3180.46 1700 7382.72 -9958.05
OPLS 2780.55 2100 -nan -11542.6

Angle Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 3616.97 230 -nan -1295.57
CHARMM25 5018.38 490 1646.81 -2783.35
OPLS 3271.23 340 -nan -1889.98

Potential Analysis

Force Field Average Err. Est. RMSD Tot-Drift (kJ/mol)
AMBER03 2.67001e+07 2.6e+07 -nan -1.60382e+08
CHARMM25 487.479 97199.742 673.043
OPLS 2.38353e+07 2.4e+07 -nan -1.39932e+08

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 (kJ/mol)
Bond 2775.97 2000 6761.45 -11375.2
Angle 3682.24 300 670.885 -1625.24
Potential 5.96e+06 5.0e+06 8.02e+07 -3.61e+07


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3186.75 2300 -nan -13603.2
Angle 3831.06 370 -nan -2070.89
Potential 3.41e+07 3.3e+07 -nan -2.03e+08


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 (kJ/mol)
Bond 3214.03 2300 7364.47 -13490.1
Angle 3738.44 310 698.943 -1792.01
Potential 7.29e+06 6.9e+06 8.86e+07 -4.38e+07


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 2341.69 1600 6048.14 -9087.07
Angle 3597.89 240 594.267 -1309.54
Potential 4.68e+06 4.7e+06 7.12e+07 -2.85e+07


Energy Average Err.Est RMSD Tot-Drift (kJ/mol)
Bond 3141.2 2300 -nan -13216.1
Angle 3672.66 290 -nan -1550.04
Potential 8.33e+06 8.1e+06 -nan -4.94e+07


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