Difference between revisions of "Task 7: MSUD - Structure-based mutation analysis"

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{|class="wikitable" border="1" style="border-spacing:0;text-align: right;"
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_b
 
  +
|Pdb
1u5b_A_1.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
 
  +
|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_b
  +
|-
  +
|1u5b_A_1.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_2.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_2.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_3.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_3.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

Revision as of 12:07, 25 June 2012

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

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
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

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_b
1u5b_A_1.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_2.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_3.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_4.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_5.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_6.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_7.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_8.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_9.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_10.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

References

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