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 had to build a runscript. We create two different scripts one for the wildtype and one for the mutations.

Additionally we had to create one file with the PDB-ID (foldx_protein.txt). And for the mutation-script a file where the mutation is declared (mutant_file.txt).

wildtype

<TITLE>FOLDX_runscript;
<JOBSTART>#;
<PDBS>#;
<BATCH>foldx_protein.txt;
<COMMANDS>FOLDX_commandfile;
<Stability>wildtype.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>#;

mutation

<TITLE>FOLDX_runscript;
<JOBSTART>#;
<PDBS>#;
<BATCH>foldx_protein.txt;
<COMMANDS>FOLDX_commandfile;
<BuildModel>#,mutant_file.txt;
<END>#;
<OPTIONS>FOLDX_optionfile;
<Temperature>298;
<R>#;
<pH>7;
<IonStrength>0.050;
<water>-CRYSTAL;
<metal>-CRYSTAL;
<VdWDesign>2;
<OutPDB>false;
<pdb_hydrogens>false;
<complex_with_DNA> true;
<END>#;
<JOBEND>#;
<ENDFILE>#;

Gromacs

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.

4.pdb2gmx

Warning: Starting residue K456 in chain not identified as Protein/RNA/DNA. Warning: Starting residue MN458 in chain not identified as Protein/RNA/DNA. Warning: Starting residue TDP556 in chain not identified as Protein/RNA/DNA.

und später dann: Fatal error: Residue 'MN' not found in residue topology database

removed first HAtoms: K, MN and TDP -> worked

6.grompp

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

processing coordinates... double-checking input for internal consistency... Reading position restraint coords from bckdha.gro renumbering atomtypes...

GB parameter(s) missing or negative for atom type 'OW'

GB parameter(s) missing or negative for atom type 'HW'

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Program grompp, VERSION 4.5.3 Source code file: /build/buildd/gromacs-4.5.3/src/kernel/grompp.c, line: 1123

Fatal error: Can't do GB electrostatics; the implicit_genborn_params section of the forcefield is missing parameters for 2 atomtypes or they might be negative. For more information and tips for troubleshooting, please check the GROMACS website at http://www.gromacs.org/Documentation/Errors

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8.Analyzation

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go back to Task 6 Sequence based mutation analysis

go to Reference Sequence BCKDHA