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

From Bioinformatikpedia
(Preparation)
(1. Choose a structure to work with)
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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 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.
+
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"/>).
   
 
<figtable id="structure_choice">
 
<figtable id="structure_choice">
Line 57: Line 57:
 
</figtable>
 
</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.
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:
 
   
  +
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 they were resolved, are too low (4.5, 5.5 and 4.6, respectively).
REMARK 465 MISSING RESIDUES <br/>
 
  +
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE <br/>
 
  +
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).
  +
<!-- REMARK 465 MISSING RESIDUES <br/>
  +
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE <br/>
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN <br/>
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN <br/>
 
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.) <br/>
 
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.) <br/>
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REMARK 465 ASP B 501 <br/>
 
REMARK 465 ASP B 501 <br/>
 
REMARK 465 THR B 502 <br/>
 
REMARK 465 THR B 502 <br/>
REMARK 465 MET B 503 <br/>
+
REMARK 465 MET B 503 <br/> -->
   
The next best candidate, '''2V3D''', has 9 residue gaps in chain A (one more than 2V3F) and 8 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.
  +
<!-- 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/>
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN <br/>
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN <br/>
Line 112: Line 114:
 
REMARK 465 ASP B 501 <br/>
 
REMARK 465 ASP B 501 <br/>
 
REMARK 465 THR B 502 <br/>
 
REMARK 465 THR B 502 <br/>
REMARK 465 MET B 503 <br/>
+
REMARK 465 MET B 503 <br/> -->
   
  +
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.
The structures '''3GXI''', '''2NT0''' and our familiar reference structure, '''1OGS''', have no missing residues.
 
  +
<!-- REMARK 465 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 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
 
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
Line 138: Line 136:
 
REMARK 465 ASP B 501
 
REMARK 465 ASP B 501
 
REMARK 465 THR B 502
 
REMARK 465 THR B 502
REMARK 465 MET B 503
+
REMARK 465 MET B 503 -->
  +
As it is the lowest resolution structure resolved at a physiological pH-value and the chain B has no gaps, we choose '''2V3E, chain B''' for this task.
 
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 18:41, 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 - 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 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"/>).

<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 five 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.

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 they 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 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. As it is the lowest resolution structure resolved at a physiological pH-value and the chain B has no gaps, 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