Structure-based mutation analysis BCKDHA

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

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 proteins have a higher energy than the unmutated protein which means that these proteins are less stable.

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 useful to save the output in a file because it includes the energy.

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

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	270ß	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
Bond
Angle
Potential

Y166N

Energy	Average	Err.Est	RMSD	Tot-Drift
Bond
Angle
Potential

G249S

Energy	Average	Err.Est	RMSD	Tot-Drift
Bond
Angle
Potential

C264W

Energy	Average	Err.Est	RMSD	Tot-Drift
Bond
Angle
Potential

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
Bond
Angle
Potential

F409C

Energy	Average	Err.Est	RMSD	Tot-Drift
Bond
Angle
Potential

Y438N

Energy	Average	Err.Est	RMSD	Tot-Drift
Bond
Angle
Potential

Links

go back to Maple syrup urine disease main page

go back to Task 6 Sequence based mutation analysis

go to Reference Sequence BCKDHA