Difference between revisions of "Task 7: MSUD - Structure-based mutation analysis"
(→MDP - file creation) |
(→foldX) |
||
Line 99: | Line 99: | ||
| Entropy Complex |
| Entropy Complex |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_N71S.pdb |
| 0.47 |
| 0.47 |
||
| 0.13 |
| 0.13 |
||
Line 123: | Line 123: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_M82I.pdb |
| -0.12 |
| -0.12 |
||
| -0.00 |
| -0.00 |
||
Line 147: | Line 147: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_Q125E.pdb |
| 1.50 |
| 1.50 |
||
| -0.01 |
| -0.01 |
||
Line 171: | Line 171: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_I213T.pdb |
| 2.21 |
| 2.21 |
||
| -0.38 |
| -0.38 |
||
Line 195: | Line 195: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_C258Y.pdb |
| 4.55 |
| 4.55 |
||
| 0.01 |
| 0.01 |
||
Line 219: | Line 219: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_T310R.pdb |
| 8.51 |
| 8.51 |
||
| 0.02 |
| 0.02 |
||
Line 243: | Line 243: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_A328T.pdb |
| 2.77 |
| 2.77 |
||
| -0.40 |
| -0.40 |
||
Line 267: | Line 267: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_I361V.pdb |
| 0.50 |
| 0.50 |
||
| 0.01 |
| 0.01 |
||
Line 291: | Line 291: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_N404S.pdb |
| 0.20 |
| 0.20 |
||
| 0.01 |
| 0.01 |
||
Line 315: | Line 315: | ||
| 0.00 |
| 0.00 |
||
|- |
|- |
||
− | | |
+ | | 1u5b_A_R429H.pdb |
| -0.08 |
| -0.08 |
||
| -0.14 |
| -0.14 |
Revision as of 12:26, 25 June 2012
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 |
A new Problem turned out here, all of the x-Ray structures contain gaps, which some of the tools can't handle. As the ph-values as well as the R-values do not differ too much, we have decided to use the structure with the least gaps: 1U5B. 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
Pdb | total energy | Backbone Hbond | Sidechain Hbond | Van der Waals | Electrostatics | Solvation Polar | Solvation Hydrophobic | Van der Waals clashes | entropy sidechain | entropy mainchain | sloop_entropy | mloop_entropy | cis_bond | torsional clash | backbone clash | helix dipole | water bridge | disulfide | electrostatic kon | partial covalent bonds | energy Ionisation | Entropy Complex |
1u5b_A_N71S.pdb | 0.47 | 0.13 | 0.13 | 0.12 | 0.00 | -0.16 | 0.21 | 0.00 | -0.16 | 0.20 | 0.00 | 0.00 | 0.00 | -0.00 | -0.02 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_M82I.pdb | -0.12 | -0.00 | 0.00 | 0.33 | -0.03 | -0.15 | 0.24 | 0.06 | -0.31 | -0.39 | 0.00 | 0.00 | 0.00 | 0.12 | 0.04 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_Q125E.pdb | 1.50 | -0.01 | 3.18 | -0.05 | -0.70 | -0.04 | -0.05 | 0.15 | -1.58 | 0.07 | 0.00 | 0.00 | 0.00 | -0.03 | -0.03 | 0.55 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_I213T.pdb | 2.21 | -0.38 | -0.49 | 1.04 | 0.00 | 0.44 | 2.54 | -0.08 | -0.49 | -0.38 | 0.00 | 0.00 | 0.00 | 0.01 | -0.44 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_C258Y.pdb | 4.55 | 0.01 | 0.17 | -1.78 | 0.07 | 1.11 | -2.84 | 7.22 | 0.31 | 0.01 | 0.00 | 0.00 | 0.00 | 0.27 | 0.90 | -0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_T310R.pdb | 8.51 | 0.02 | 0.28 | -1.67 | 0.13 | 3.01 | -1.29 | 4.89 | 1.30 | 0.04 | 0.00 | 0.00 | 0.00 | 2.22 | 0.11 | -0.42 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_A328T.pdb | 2.77 | -0.40 | -0.41 | -0.63 | -0.01 | 1.14 | -0.63 | 2.76 | 0.51 | -0.92 | 0.00 | 0.00 | 0.00 | 1.36 | -0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_I361V.pdb | 0.50 | 0.01 | -0.01 | 0.48 | 0.00 | -0.28 | 0.86 | -0.04 | -0.55 | 0.02 | 0.00 | 0.00 | 0.00 | 0.01 | -0.07 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | -0.01 | 0.00 |
1u5b_A_N404S.pdb | 0.20 | 0.01 | 0.20 | 0.37 | 0.00 | -0.48 | 0.50 | -0.02 | -0.25 | -0.22 | 0.00 | 0.00 | 0.00 | 0.09 | -0.11 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
1u5b_A_R429H.pdb | -0.08 | -0.14 | -0.19 | 0.37 | 0.14 | -0.50 | 0.40 | 0.03 | -0.43 | 0.26 | 0.00 | 0.00 | 0.00 | -0.19 | -0.20 | -0.07 | 0.00 | 0.00 | 0.00 | 0.00 | 0.25 | 0.00 |
Minimise
Minimise entry will be here soonish
Gromacs
Step 1 fetch-pdb
1.Use fetchpdb to get the pdb structure – look at the script, what does it do?
This script in meant to fetch pdb-files from the server. After a check was passed if the given pdb-file-name is valid, the archive is downloaded, extracted and everything but the pdb-file is removed. In our case fetch-pdb was not used, because we had to use our prepared pdb-file without the gaps in it.
repair pdb
2.Use repairPDB to clean the PDB and extract the protein only - describe what options you chose and what other options are available. Make sure you chose the right chain.
To extract the protein without side chains and solvent the options -noh and -nosol from repair pdb were used.
repairPDB 1u5b.pdb -noh -cleansol > 1u5b_noside.pdb
A complete list of parameters of repairPDB can be found in the journal.
SCWRL
For SCWRL the sequence had to be parsed out of the new pdb-file:
repairPDB 1u5b_noside.pdb -seq > 1u5b_sequence
All aminoacids were turned into small letters, and then SCWRL was run:
/opt/SS12-Practical/scwrl4/Scwrl4 -i 1u5b_clean.pdb -s 1u5b-sequence -o 1u5b_scwrl.pdb
After SCWRL, the hydrogens had to be removed again:
repairPDB 1u5b_scwrl.pdb -noh -cleansol > 1u5b_scwrl_noside.pdb
pdb2gmx
We have chosen to use the model amber03 for the first forcefield.
/opt/SS12-Practical/gromacs/bin/pdb2gmx -f 1u5b_scwrl_noside.pdb -o 1u5b_gromacs.out -p 1u5b_gromacs.top -water tip3p -ff amber03
MDP - file creation
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
grompp
/opt/SS12-Practical/gromacs/bin/grompp -v -f config.mdp -c 1u5b_gromacs.out.gro -p 1u5b_gromacs.top -o 1u5b.tpr
mdrun
/opt/SS12-Practical/gromacs/bin/mdrun -v -deffnm 1u5b.tpr
analysis
/opt/SS12-Practical/gromacs/bin/g_energy -f 1u5b.tpr.edr -o energy1.xvg
References
<references />