Structure-based mutation analysis BCKDHA
Contents
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
SCWRL
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 |
foldX
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.
<TITLE>FOLDX_runscript; <JOBSTART>#; <PDBS>#; <BATCH>foldx_protein.txt; <COMMANDS>FOLDX_commandfile; <Stability>list.txt; <END>#; <OPTIONS>FOLDX_optionfile; //<Temperature>298; <R>#; <pH>5.5; <IonStrength>0.050; <water>-CRYSTAL; <metal>-CRYSTAL; <VdWDesign>2; <OutPDB>false; <pdb_hydrogens>false; <END>#; <JOBEND>#; <ENDFILE>#;
| 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 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.
Minimise
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 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 |
| mutant1 | -4253.174790 | 1767.722015 |
| mutant2 | -4080.989512 | 1595.536757 |
| mutant3 | -4354.495238 | 1869.042483 |
| mutant4 | -4280.043000 | 1794.590245 |
| mutant5 | -3745.313620 | 1259.860865 |
| mutant6 | -3989.790625 | 1504.33787 |
| mutant7 | -4317.105618 | 1831.652863 |
| mutant8 | -4358.528143 | 1873.075388 |
| mutant9 | -4339.778964 | 1854.326209 |
gromacs
Gromacs
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.
General
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
3. SCWRL
scwrl -i bckdha_mod.pdb -s extractedPDB.seq -o bckdha_scwrl.pdb
pdb including HEATOMS
4.pdb2gmx
use clean pdb without HEATOMS
pdb2gmx -f bckdha_clean.pdb -o bckdha.gro -p bckdha.top -water tip3p -ff amber03
5. MDP
6. grompp
grompp -v -f MDP_bckdha.mdp -c bckdha.gro -p bckdha.top -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
Analysis
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
- OPLS-AA
Mutation analysis
M82L
| 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 |
Q125E
| 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 |
Y166N
| 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 |
G249S
| 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 |
C264W
| 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 |
R265W
| 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 |
I326T
| 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 |
F409C
| 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 |
Y438N
| 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 |
Links
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go back to Task 6 Sequence based mutation analysis
