Difference between revisions of "Gaucher Disease: Task 09 - Lab Journal"
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Before minimise the hydrogens and waters (protein only) need to be removed using repairPDB. |
Before minimise the hydrogens and waters (protein only) need to be removed using repairPDB. |
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+ | Minimise can be executed with the following command: |
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− | Minimise call: |
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− | minimise <input structure |
+ | /opt/SS12-Practical/minimise/minimise <input structure file> <output structure file> <log file> |
Revision as of 00:13, 31 August 2013
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This page is under construction.
Contents
Preparation
1. Choice of a structure to work with
In the former tasks, we worked with the reference structure 1OGS, because it has no gaps - except for an offset of 39 residues at the N terminus that all structures for our protein P04062 referenced in UniProt have - and has a pretty high resolution of 2.0 Å. However, there are five other structures with a higher or equal resolution (all resolved using the X-ray diffraction method). We compared 1OGS and those five structures for the resolution, coverage and gaps, R-factor, R-free and pH-value at which the structure was resolved in order to choose the best structure suiting the requirements of this task (<xr id="structure_choice"/>).
<figtable id="structure_choice">
PDB-ID | Resolution (Å) | Chain | Covered residues (UniProt seq.) | Missing residues (ATOM seq.) | Covered residues (ATOM seq.) | R-Value(obs.) | R-Free | pH | Temperature (K) |
---|---|---|---|---|---|---|---|---|---|
2NT0 | 1.79 | A/B/C/D | 40-536 (92.7%) | - | 1-497 | 0.181 | 0.215 | 4.5 | 100 |
3GXI | 1.84 | A/B/C/D | 40-536 (92.7%) | - | 1-497 | 0.193 | 0.231 | 5.5 | NULL |
2V3F | 1.95 | A/B | 40-536 (92.7%) | A: 29-31, (499-503), B: (-1-0), 27-32, (498-503) | A: -1-28, 32-498, B: 1-26, 33-497 | 0.154 | 0.196 | 6.5 | 100 |
2V3D | 1.96 | A/B | 40-536 (92.7%) | A: 28-31, (499-503), B: (-1-0), (498-503) | A: -1-27, 32-498, B: 1-497 | 0.157 | 0.208 | 6.5 | 100 |
2V3E | 2.0 | A/B | 40-536 (92.7%) | A: 31, (498-503), B: (-1), (498-503) | A: -1-30, 32-497, B: 0-497 | 0.163 | 0.220 | 7.5 | 100 |
1OGS | 2.0 | A/B | 40-536 (92.7%) | - | 1-497 | 0.195 | 0.230 | 4.6 | 100 |
</figtable>
In all the investigated structures residues numbered 1-497 are native and correspond to the positions 40-536 in the UniProt sequence P04062. In some of the structures, like in 2V3E, two residues were added at the beginning (EF, positions -1, 0) and six residues at the end (LLVDTM, positions 498-503). If these "axillary" residues are missing in the resolved structure, we can ignore it. For this reason they are presented in brackets in the table. To be sure, we verified that the PDB annotations about the missing residues are correct using a simple python script.
The structures 2NT0, 3GXI and our familiar reference structure, 1OGS, have no missing residues and the first two candidates have the highest resolutions (1.79 and 1.84, respectively). However, the pH-values, at which the structures were resolved, are too low (4.5, 5.5 and 4.6, respectively).
The structure 2V3F seems to be a good choice, because it compromises between the pH-value (pH=6.5 is near to the physiological value of 7.4) and high resolution (1.95 Å). Moreover, it has the lowest R-value (0.154) and R-free (0.196). However, there are some missing residues (29-31 in chain A and 27-32 in chain B).
The candidate 2V3D has similar values, but it also has gaps in chain A (28-31). We could have used the chain B, thought.
A structure candidate with the same resolution as 1OGS and a neutral pH of 7.5 is 2V3E. It has only one missing residue in chain A (31) and none native residues are missing in chain B. Moreover, this structure has relatively low R-value (0.163) and R-free (0.220) values, which are lower than in 2NT0, 3GXI or 1OGS. As 2V3E is the highest resolution structure resolved at a physiological pH-value and the chain B has no gaps, we choose 2V3E, chain B.
2. Visualization of the mutations to work with
We prepared a PDB structure of 2V3E, chain B, without the axillary residue number 0 with PyMol, with which we also visualized the residues to be mutated on this structure.
3. Creation of mutated structures
We ran SCWRL as follows:
/opt/SS12-Practical/scwrl4/Scwrl4
-i <backbone_IN.pdb> input PDB file of the backbone (of 2V3E_B without the residue number 0, created in PyMol)
-o <model_OUT.pdb> output PDB file of the model
-s <sequence file> file with ATOM sequence of the model file in lower case with one mutated residue we want to predict in uppercase
-0 normal priority mode
We prepared the sequence files as follows. We downloaded the FASTA sequence of 2V3E_B from the PDB, deleted the header and the newlines. Then, we deleted the first two and the last six additional residues mentioned before. This is the sequence of the actually resolved structure. Next, we changed all letters to lower case (with VIM editor). Finally, for each mutation, we introduced the new amino acid letter in capital letter.
Energy comparisons
foldX
We used the approach "Multiple mutations using individual list" with the option 1: "-manual", as described in the examples page and foldX manual. The version 3.0 Beta 5.1 was used.
Minimise
Before minimise the hydrogens and waters (protein only) need to be removed using repairPDB.
Minimise can be executed with the following command:
/opt/SS12-Practical/minimise/minimise <input structure file> <output structure file> <log file>
Sources
PDB R-value and R-free
FoldX:
1) Francois Stricher, Tom Lenaerts, Joost Schymkowitz, Frederic Rousseau and Luis Serrano (2008). FoldX 3.0. “In preparation”
2) Schymkowitz J., Borg J., Stricher F., Nys R., Rousseau F., Serrano L. (2005). The FoldX web server: an online force field. Nucleic Acids Research, vol 33, pW382-8.
3) Schymkowitz J. W., Rousseau F., Martins I. C., Ferkinghoff-Borg J., Stricher F., Serrano L. (2005). Prediction of water and metal binding sites and their affinities by using the Fold-X force field. Proc Natl Acad Sci USA, vol 102, p 10147-52.
4) Guerois R., Nielsen J. E., Serrano L. (2002). Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol, vol 320, p369-87.