Task 7: MSUD - Structure-based mutation analysis

From Bioinformatikpedia
Revision as of 18:05, 24 June 2012 by Wagnerr (talk | contribs) (minimize)

Task description

preparation

First an x-Ray pdb structure<ref>http://www.uniprot.org/uniprot/P12694</ref> file had to be chosen for our protein.

It is important that the structure has a high resolution (small Å value);
furthermore the R-factor should be as small as possible, and the higher the coverage the better.
Also, check at which pH-value the structure was resolved; ideally you want physiological pH (7.4).
EntryMethodResolution(Å)ChainPositions
1DTWX-ray2.70A46-445
1OLSX-ray1.85A46-445
1OLUX-ray1.90A46-445
1OLXX-ray2.25A46-445
1U5BX-ray1.83A46-445
1V11X-ray1.95A46-445
1V16X-ray1.90A46-445
1V1MX-ray2.00A46-445
1V1RX-ray1.80A46-445
1WCIX-ray1.84A46-445
1X7WX-ray1.73A46-445
1X7XX-ray2.10A46-445
1X7YX-ray1.57A46-445
1X7ZX-ray1.72A46-445
1X80X-ray2.00A46-445
2BEUX-ray1.89A46-445
2BEVX-ray1.80A46-445
2BEWX-ray1.79A46-445
2BFBX-ray1.77A46-445
2BFCX-ray1.64A46-445
2BFDX-ray1.39A46-445
2BFEX-ray1.69A46-445
2BFFX-ray1.46A46-445
2J9FX-ray1.88A/C46-445

We first looked at the structure with the best resolution: 2BFD. It contains chain A and B of the protein. For us only chain A is of interest, here the file covers 400 residues( 90% of the uniprot sequence ). The structure was generated the 12th of april in 2004, using a PH-value of 5.5. Although these values would be ok, we compared them to the next best structures( looking at the resolution ):

pdbresolution(A°)phR-value
2BFD1.395.50.150
2BFF1.465.50.150
1X7Y1.575.80.150
2BFC1.645.50.144
2BFE1.695.50.150
1U5B1.835.80.156

As the ph-values as well as the R-values do not differ too much, we have decided to use the structure with the best resolution: 2BFD. As only chain A is of interest, chain B as well as all gaps at the end of chain A were removed using pymol. The next step was, to prepare our list of SNP's, and substitute those, that are not contained in the pdb-sequence. All pdb-files cover position 46-445. This means we had to substitute the SNP L17F. L17F is not listed in the HGMD, and for this seen as neutral, what means we had to chose another neutral SNP for it. We've chosen A71G. The new list of SNP's then is:

N71S, M82I, Q125E, I213T, C258Y, T310R, A328T, I361V, N404S, R429H
The picture shows the 10 SNP's colored in blue on the chain A of obda_human in green. The structure is shown with pymol using the modified PDB file.

obda_human has 4 annotated sites in the uniprot entry P12694:

posfunctionsnp
157-159Thiamine pyrophosphate binding -
206metal binding -
211metal binding -
212metal binding -

tools

SCWRL

It is possible to give SCWRL the mutated sequence.
This can be done by extracting the sequence with repairPDB.
Then you change all letters to lower case. Next you introduce the new amino acid letter (mutation) in capital letters to the sequence file.
This sequence file can be read in by SCWRL using the –s flag. Check if only the mutation side chain has been changed.

the extraction of the sequence and a mapping of positions between snps and pdb-sequence can be found in the journal

foldX

FoldX entry will be here soonish

Minimise

Minimise entry will be here soonish

gromacs

gromacs entry will be here soonish

References

<references />