Difference between revisions of "Gaucher Disease: Task 09 - Lab Journal"

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(foldX)
(1. Choice of a structure to work with)
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===1. Choice of a structure to work with===
 
===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 [http://www.uniprot.org/uniprot/P04062 UniProt] have - and has a pretty low resolution of 2.0 Å. However, there are five other structures with a lower 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"/>).
+
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 [http://www.uniprot.org/uniprot/P04062 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">
 
<figtable id="structure_choice">
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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.
 
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 lowest 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 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 low 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 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).
 
<!-- REMARK 465 MISSING RESIDUES <br/>
 
<!-- REMARK 465 MISSING RESIDUES <br/>
 
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE <br/>
 
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE <br/>
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REMARK 465 THR B 502
 
REMARK 465 THR B 502
 
REMARK 465 MET B 503 -->
 
REMARK 465 MET B 503 -->
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 lowest resolution structure resolved at a physiological pH-value and the chain B has no gaps, we choose '''2V3E, 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===
 
===2. Visualization of the mutations to work with===

Revision as of 22:44, 30 August 2013

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This page is under construction.

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
Comparison of the resolution top six PDB structures according to different other criteria.

</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 indivudal list, as described in the examples page.

Minimise

Gromacs (optional task for those who love MD!)

Sources

PDB R-value and R-free