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

From Bioinformatikpedia
(Preparation)
(Preparation)
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===1. Choose a structure to work with===
 
===1. Choose a structure to work with===
   
In the former tasks, we worked with the reference structure 1OGS, because it has no gaps - but an offset of 39 residues at the N terminus, as all structures for our protein P04062 referenced in [http://www.uniprot.org/uniprot/P04062 UniProt] - and has a pretty low resolution of 2.0 Å. However, there are four other structures with a lower resolution (all resolved using the X-ray diffraction method). We compare 1OGS and those four structures for the resolution, coverage and gaps, R-factor, R-free and pH-value at which the structure was resolved in the following table.
+
In the former tasks, we worked with the reference structure 1OGS, because it has no gaps - but an offset of 39 residues at the N terminus, as all structures for our protein P04062 referenced in [http://www.uniprot.org/uniprot/P04062 UniProt] - 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 compare 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 the following table.
   
 
<figtable id="structure_choice">
 
<figtable id="structure_choice">
Line 57: Line 57:
 
</figtable>
 
</figtable>
   
The structure '''2V3F''' seems to be a good choice because of the highest pH-value and the lowest R-value and R-free. However, there are some missing residues, 8 in chain A:
+
The structure '''2V3F''' seems to be a good choice because of the compromise between the pH-value and the lowest R-value and R-free. However, there are some missing residues, 8 in chain A:
   
 
REMARK 465 MISSING RESIDUES <br/>
 
REMARK 465 MISSING RESIDUES <br/>
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REMARK 465 MET B 503 <br/>
 
REMARK 465 MET B 503 <br/>
   
The structures '''3GXI''', '''2NT0''' and our familiar reference structure, '''1OGS''', has no missing residues (no comment, TODO: check!).
+
The structures '''3GXI''', '''2NT0''' and our familiar reference structure, '''1OGS''', have no missing residues.
  +
  +
A structure candidate with the same resolutuin as 1OGS and a neutral pH of 7.5 is '''2V3E''', however it has some missing residues in chain A
  +
and B:
  +
  +
REMARK 465 MISSING RESIDUES
  +
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
  +
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
  +
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
  +
REMARK 465
  +
REMARK 465 M RES C SSSEQI
  +
REMARK 465 PHE A 31
  +
REMARK 465 LEU A 498
  +
REMARK 465 LEU A 499
  +
REMARK 465 VAL A 500
  +
REMARK 465 ASP A 501
  +
REMARK 465 THR A 502
  +
REMARK 465 MET A 503
  +
REMARK 465 GLU B -1
  +
REMARK 465 LEU B 498
  +
REMARK 465 LEU B 499
  +
REMARK 465 VAL B 500
  +
REMARK 465 ASP B 501
  +
REMARK 465 THR B 502
  +
REMARK 465 MET B 503
  +
  +
Nevertheless, all the missing residues in chain B are not included in the gapless structures we inverstigated anyway (and most of the
  +
missing residues in chain A, only the residue 31 is really a gap). In all the inverstigated structures residues numbers 1-497 are
  +
native and correspond to the positions 40-536 in the UniProt sequence P04062. In some structures of Glucocerobrosidase, like in 2V3E,
  +
residues EF at the beginning (-1, 0) and residues LLVDTM at the end (498-503) were added. Therefore, all the missing residues in the
  +
chain B of 2V3E are not native and can be ignored. As it is the lowest resolution structure resolved at a physiological pH-value
  +
and the chains A and B are identical, we choose '''2V3E, chain B''' for this task.
   
 
===2. Visualise the mutations you want to work with===
 
===2. Visualise the mutations you want to work with===

Revision as of 15:23, 29 August 2013

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This page is under construction.

Preparation

1. Choose a structure to work with

In the former tasks, we worked with the reference structure 1OGS, because it has no gaps - but an offset of 39 residues at the N terminus, as all structures for our protein P04062 referenced in UniProt - 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 compare 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 the following table.

<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 ?
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 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 five PDB structures according to different other criteria.

</figtable>

The structure 2V3F seems to be a good choice because of the compromise between the pH-value and the lowest R-value and R-free. However, there are some missing residues, 8 in chain A:

REMARK 465 MISSING RESIDUES
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
REMARK 465
REMARK 465 M RES C SSSEQI
REMARK 465 PRO A 29
REMARK 465 THR A 30
REMARK 465 PHE A 31
REMARK 465 LEU A 499
REMARK 465 VAL A 500
REMARK 465 ASP A 501
REMARK 465 THR A 502
REMARK 465 MET A 503
REMARK 465 GLU B -1
REMARK 465 PHE B 0
REMARK 465 ASP B 27
REMARK 465 PRO B 28
REMARK 465 PRO B 29
REMARK 465 THR B 30
REMARK 465 PHE B 31
REMARK 465 PRO B 32
REMARK 465 LEU B 498
REMARK 465 LEU B 499
REMARK 465 VAL B 500
REMARK 465 ASP B 501
REMARK 465 THR B 502
REMARK 465 MET B 503

The next best candidate, 2V3D, has 9 residue gaps in chain A (one more than 2V3F) and 8 in chain B:

REMARK 465 MISSING RESIDUES
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
REMARK 465
REMARK 465 M RES C SSSEQI
REMARK 465 PRO A 28
REMARK 465 PRO A 29
REMARK 465 THR A 30
REMARK 465 PHE A 31
REMARK 465 LEU A 499
REMARK 465 VAL A 500
REMARK 465 ASP A 501
REMARK 465 THR A 502
REMARK 465 MET A 503
REMARK 465 GLU B -1
REMARK 465 PHE B 0
REMARK 465 LEU B 498
REMARK 465 LEU B 499
REMARK 465 VAL B 500
REMARK 465 ASP B 501
REMARK 465 THR B 502
REMARK 465 MET B 503

The structures 3GXI, 2NT0 and our familiar reference structure, 1OGS, have no missing residues.

A structure candidate with the same resolutuin as 1OGS and a neutral pH of 7.5 is 2V3E, however it has some missing residues in chain A and B:

REMARK 465 MISSING RESIDUES REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.) REMARK 465 REMARK 465 M RES C SSSEQI REMARK 465 PHE A 31 REMARK 465 LEU A 498 REMARK 465 LEU A 499 REMARK 465 VAL A 500 REMARK 465 ASP A 501 REMARK 465 THR A 502 REMARK 465 MET A 503 REMARK 465 GLU B -1 REMARK 465 LEU B 498 REMARK 465 LEU B 499 REMARK 465 VAL B 500 REMARK 465 ASP B 501 REMARK 465 THR B 502 REMARK 465 MET B 503

Nevertheless, all the missing residues in chain B are not included in the gapless structures we inverstigated anyway (and most of the missing residues in chain A, only the residue 31 is really a gap). In all the inverstigated structures residues numbers 1-497 are native and correspond to the positions 40-536 in the UniProt sequence P04062. In some structures of Glucocerobrosidase, like in 2V3E, residues EF at the beginning (-1, 0) and residues LLVDTM at the end (498-503) were added. Therefore, all the missing residues in the chain B of 2V3E are not native and can be ignored. As it is the lowest resolution structure resolved at a physiological pH-value and the chains A and B are identical, we choose 2V3E, chain B for this task.

2. Visualise the mutations you want to work with

3. Create mutated structures

Energy comparisons

foldX

Minimise

Gromacs (optional task for those who love MD!)

Sources

PDB R-value and R-free