Task 7: MSUD - Structure-based mutation analysis
Contents
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).
Entry | Method | Resolution(Å) | Chain | Positions |
1DTW | X-ray | 2.70 | A | 46-445 |
1OLS | X-ray | 1.85 | A | 46-445 |
1OLU | X-ray | 1.90 | A | 46-445 |
1OLX | X-ray | 2.25 | A | 46-445 |
1U5B | X-ray | 1.83 | A | 46-445 |
1V11 | X-ray | 1.95 | A | 46-445 |
1V16 | X-ray | 1.90 | A | 46-445 |
1V1M | X-ray | 2.00 | A | 46-445 |
1V1R | X-ray | 1.80 | A | 46-445 |
1WCI | X-ray | 1.84 | A | 46-445 |
1X7W | X-ray | 1.73 | A | 46-445 |
1X7X | X-ray | 2.10 | A | 46-445 |
1X7Y | X-ray | 1.57 | A | 46-445 |
1X7Z | X-ray | 1.72 | A | 46-445 |
1X80 | X-ray | 2.00 | A | 46-445 |
2BEU | X-ray | 1.89 | A | 46-445 |
2BEV | X-ray | 1.80 | A | 46-445 |
2BEW | X-ray | 1.79 | A | 46-445 |
2BFB | X-ray | 1.77 | A | 46-445 |
2BFC | X-ray | 1.64 | A | 46-445 |
2BFD | X-ray | 1.39 | A | 46-445 |
2BFE | X-ray | 1.69 | A | 46-445 |
2BFF | X-ray | 1.46 | A | 46-445 |
2J9F | X-ray | 1.88 | A/C | 46-445 |
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 ):
pdb | resolution(A°) | ph | R-value |
2BFD | 1.39 | 5.5 | 0.150 |
2BFF | 1.46 | 5.5 | 0.150 |
1X7Y | 1.57 | 5.8 | 0.150 |
2BFC | 1.64 | 5.5 | 0.144 |
2BFE | 1.69 | 5.5 | 0.150 |
1U5B | 1.83 | 5.8 | 0.156 |
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
obda_human has 4 annotated sites in the uniprot entry P12694:
pos | function | snp |
157-159 | Thiamine pyrophosphate binding | - |
206 | metal binding | - |
211 | metal binding | - |
212 | metal 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
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