Structure-based mutation analysis BCKDHA
Contents
Structure selection
In order to perform a structure based mutation analysis an appropriate protein structure is needed. Therefore one has to check whether the resolution of the structure is high enough, that means the Å-value should be small. The R-factor (reliability factor or residual factor) is another measure to access the quality of a structure. The R-factor rates how well the crystallographic model predicts the observed data from experimental X-ray diffraction. The smaller the R-factor the better. The coverage is the percentage of UniProt sequence present in the sample structure. This value was obtained from [[1]]. The coverage should be quite high. The pH-value indicates the pH-value at which the structure was resolved. Ideally it should be resolved at physiological pH (7.4).
The following table presents the PDB structures for BCKDHA to date. For each structure the resolution in [Å], the R-factor, coverage and pH-value are listed as well.
| PDB id | resolution [Å] | R-factor | % of UniProt sequence present in the sample | %residues observed | ph-value |
|---|---|---|---|---|---|
| 1DTW | 2.70 | 0.224 | 90% | 97% | 7.5* |
| 1OLS | 1.85 | 0.172 | 90% | 96% | 5.5 |
| 1OLU | 1.90 | 0.161 | 90% | 91% | 5.5 |
| 1OLX | 2.25 | 0.161 | 90% | 97% | 5.5 |
| 1U5B | 1.83 | 0.156 | 90% | 97% | 5.8 |
| 1V11 | 1.95 | 0.139* | 90% | 92% | 5.5 |
| 1V16 | 1.90 | 0.132* | 90% | 92% | 5.5 |
| 1V1M | 2.00 | 0.130* | 90% | 93% | 5.5 |
| 1V1R | 1.80 | 0.158 | 90% | 88% | 5.5 |
| 1WCI | 1.84 | 0.149 | 90% | 97% | 5.5 |
| 1X7W | 1.73 | 0.148 | 90% | 92% | 5.8 |
| 1X7X | 2.10 | 0.149 | 90% | 92% | 5.8 |
| 1X7Y | 1.57 | 0.150 | 90% | 92% | 5.8 |
| 1X7Z | 1.72 | 0.154 | 90% | 97% | 5.8 |
| 1X80 | 2.00 | 0.161 | 90% | 92% | 5.8 |
| 2BEU | 1.89 | 0.171 | 90% | 97% | 5.5 |
| 2BEV | 1.80 | 0.139 | 90% | 97% | 5.5 |
| 2BEW | 1.79 | 0.147 | 90% | 97% | 5.5 |
| 2BFB | 1.77 | 0.145 | 90% | 92% | 5.5 |
| 2BFC | 1.64 | 0.144 | 90% | 92% | 5.5 |
| 2BFD | 1.39* | 0.150 | 90% | 93% | 5.5 |
| 2BFE | 1.69 | 0.150 | 90% | 93% | 5.5 |
| 2BFF | 1.46 | 0.150 | 90% | 98% | 5.5 |
| 2J9F | 1.88 | 0.171 | 90% | 95% | 5.5 |
The asteriks-marked values indicate that these structures were resolved with the asked experimental quality. As one can see, none of the structures fulfills all conditions.
Furthermode, 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 start position corresponds to position 6 of the BCKDHA -PDB sequence.
As we can see none of the values corresponds to the demands because it was asked for a 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
Figure 1 and 2 shows the structure of BCKDHA with mapped mutations.
As shown in Figure 2 there are some mutations on the surface of the protein, which might have an effect on the stability of the protein. Furthermore it is possible to recognize a mutation which is quite near to the active center. These facts are studied in more detail in the mutation analysis section.
Comparison energies
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. 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.
Minimise
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. Then it calculates the energy for the whole mutated model. The aim by comparing the mutated models with the wildtype is to find out if there is a structural change caused by a mutation. The calculated energies are discussed in more detail in the mutation analysis section (below).
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
For repairPDB the following options are available:
| -offset value | offset the residue numbering |
| -chain char | change Chain ID |
| -ratom | renumber Atoms |
| -rres | renumber Residues |
| -noh | remove hydrogens |
| -het | no change of HETATM to ATOM for AA |
| -seq | returns protein sequence from AA in pdb file |
| -seqrs | protein sequence from SEQRES entries |
| -nosol | just Protein, no solvent OR |
| -ssw cutoff | print only waters with B-value below cutoff OR |
| -cleansol | remove overlapping solvent for GROMACS |
We run repairPDB using the following command:
repairPDB bckdha_mod.pdb -noh -nosol > bckdha_clean.pdb
Using this command we removed hydrogens and solvent from our pdb to get just the protein.
3. SCWRL
SCWRL was executed using the following command:
scwrl -i bckdha_mod.pdb -s extractedPDB.seq -o bckdha_scwrl.pdb
SCWRL returned a pdb including HETATOMS. These solvent atoms needed to be removed before continuing.
4.pdb2gmx
(use clean pdb without HEATOMS!)
pdb2gmx -f bckdha_clean.pdb -o bckdha.gro -p bckdha.top -water tip3p -ff amber03
5. MDP
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
adjust nsteps for the time vs steps analysis
| integrator | a steepest descent algorithm for energy minimization. |
| emtol | tolerance for steep integrator:the minimization is converged when the maximum force is smaller than this value |
| nsteps | maximum number of steps to integrate or minimize, -1 is no maximum |
| nstenergy | frequency to write energies to energy file (last energies are always written) |
| energygrps | groups to write to energy files |
| ns_type | Which atoms to check when construction a new neighbor list |
| coulombtype | which coulomb-type to use, e.g. Cut-off, PME, Ewald |
| rcoulomb | distance for the Coulomb cut-off |
| rvdw | Van-der-Waals cut-of (distance for the LJ or Buckingham cut-off) |
| constraints | constraints for bonds, e.g bonds are presented by a potention, all bonds are converted to constraints,... |
| pbc | Remove the periodicity (make molecule whole again) |
6. grompp
grompp -v -f 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
The table below shoes the running time for mdrun depending on different values for nsteps. It also lists the real number of steps carried out to calculate the energy.
| steps | time (real) [s] | time (user) [s] | time (sys) [s] | performed steps |
|---|---|---|---|---|
| 50 | 5.453 | 4.730 | 0.120 | 50 |
| 100 | 10.393 | 9.210 | 0.240 | 100 |
| 500 | 36.419 | 30.660 | 0.780 | 338 |
| 1000 | 5.261 | 4.390 | 0.130 | 47 |
| 2000 | 10.564 | 8.500 | 0.290 | 93 |
| 3000 | 10.661 | 8.840 | 0.230 | 96 |
| 4000 | 2.620 | 2.010 | 0.140 | 21 |
| 5000 | 3.693 | 3.300 | 0.100 | 35 |
Figure 3 shows the correlation between nsteps and the running time for mdrun
Interestingly, the running time is not dependent on the number of nsteps, but just on the number of really performed steps. There is a linear dependency between the calculation time and the number of performed steps. The number of performed steps however is not correlating with the value for nsteps. It is not obvious why the number of performed steps varies so extremely given a certain value for nsteps.
Wildtype analysis: force fields
The different force fields chosen for this task were:
- AMBER03
- CHARMM27
- OPLS-AA
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 | 97 | 199.742 | 673.043 |
| OPLS | 2.38353e+07 | 2.4e+07 | -nan | -1.39932e+08 |
Comparing the results for the different force fields it is noticeable that only Charmm27 produces values for the RMSD. The values calculated by AMBER03 and OPLS are mostly similar, only the CHARMM27 values vary totally.
Figure 4 shows the energy calculated by Gromacs using the AMBER03 forcefield applied to the wildtype protein structure. Figure 5 and 6 are showing the energy for the Charmm25 and the OPLS-AA force fields, respectively.
Mutation analysis
In order to discuss the effect of different mutations more in detail, one page for each mutation was created:
Results
The following table summarizes the calculated energies for the different mutations.
| FoldX | Minimise | Gromacs | ||||
| Mutation | energy value | difference | energy value | difference | energy value | difference |
| wildtype | 401.00 | 0 | -2485.452755 | 0 | 2.67001e+07 | 0 |
| M82L | 437.88 | 36.88 | -4253.174790 | -1767.722015 | 5.16e+06 | -21540100 |
| Q125E | 431.77 | 30.77 | -4080.989512 | -1595.536757 | 5.23e+06 | -21470100 |
| Y166N | 432.24 | 31.24 | -4354.495238 | -1869.042483 | 7.95e+06 | -18750100 |
| G249S | 432.22 | 31.22 | -4280.043000 | -1794.590245 | 5.96e+06 | -20740100 |
| C264W | 488.43 | 87.43 | -3745.313620 | -1259.860865 | 3.41e+07 | 7399900 |
| R265W | 460.43 | 59.43 | -3989.790625 | -1504.33787 | 5.36e+06 | -21340100 |
| I326T | 432.94 | 31.94 | -4317.105618 | -1831.652863 | 7.29e+06 | -19410100 |
| F409C | 433.33 | 32.33 | -4358.528143 | -1873.075388 | 4.68e+06 | -22020100 |
| Y438N | 431.56 | 30.56 | -4339.778964 | 1854.326209 | 8.33e+06 | -18370100 |
Interestingly, foldX calculates for all mutations a higher energy than for the wildtype, whereas the energy of the mutated protein calculated by Minimise and Gromacs is (almost) always lower than the wildtype energy. The difference in energies is also very varying between these tools. While foldX returns usually very small energy differences between wildtype and mutated structures, the differences are bigger for minimise and even higher for Gromacs. The most interesting observation from these energies is the mutation C264W, where foldX calculates a very different energy for the mutated protein and also Gromacs returns for the only time an energy that is higher for the mutated structure than for the wildtype structure. See detailed mutation analysis for more information.
Links
go back to Maple syrup urine disease main page
go back to Task 6 Sequence based mutation analysis
go to Task 8 Molecular Dynamics Simulations
