Difference between revisions of "Structure-based mutation analysis (Phenylketonuria)"

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(→‎Preparation)
(→‎Preparation)
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! style="background:#32CD32;" | Coverage %
  
! style="background:#32CD32;" | Coverage %
 
|-
  
|-
−
| 1DMW || 2.00 || 0.20 || 6.80 || x || A || 118-424 || x
 +
| 1DMW || 2.00 || 0.20 || 6.80 || 0 || A || 118-424 || x
 
|-
  
|-
−
| 1J8T || 1.70 || 0.20 || 6.80 || x || A || 103-427 || x
+
| 1J8T || 1.70 || 0.20 || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
 
| 1J8U || <span style="background:#00FF00">1.50</span> || <span style="background:#00FF00">0.16</span> || 6.80 || 0 || A || 103-427 || x
 
| 1J8U || <span style="background:#00FF00">1.50</span> || <span style="background:#00FF00">0.16</span> || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
−
| 1KW0 || 2.50 || 0.22 || 6.80 || x || A || 103-427 || x
+
| 1KW0 || 2.50 || 0.22 || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
−
| 1LRM || 2.10 || 0.21 || 6.80 || x || A || 103-427 || x
+
| 1LRM || 2.10 || 0.21 || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
−
| 1MMK || 2.00 || 0.20 || 6.80 || x || A || 103-427 || x
+
| 1MMK || 2.00 || 0.20 || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
−
| 1MMT || 2.00 || 0.21 || 6.80 || x || A || 103-427 || x
+
| 1MMT || 2.00 || 0.21 || 6.80 || 0 || A || 103-427 || x
 
|-
  
|-
−
| 1PAH || 2.00 || 0.18 || 6.80 || x || A || 117-424 || x
+
| 1PAH || 2.00 || 0.18 || 6.80 || 0 || A || 117-424 || x
 
|-
  
|-
−
| 1TDW || 2.10 || 0.21 || 6.80 || x || A || 117-424 || x
+
| 1TDW || 2.10 || 0.21 || 6.80 || aa-change: || A || 117-424 || x
 
|-
  
|-
−
| 1TG2 || 2.20 || 0.21 || 6.80 || x || A || 117-424 || x
+
| 1TG2 || 2.20 || 0.21 || 6.80 || aa-change: || A || 117-424 || x
 
|-
  
|-
−
| 2PAH || 3.10 || 0.25 || <span style="background:#00FF00">7.00</span> || x || A/B || 118-452 || x
 +
| 2PAH || 3.10 || 0.25 || <span style="background:#00FF00">7.00</span> || 0 || A/B || 118-452 || x
 
|-
  
|-
−
| 3PAH || 2.00 || 0.18 || 6.80 || x || A || 117-424 || x
+
| 3PAH || 2.00 || 0.18 || 6.80 || 0 || A || 117-424 || x
 
|-
  
|-
−
| 4ANP || 2.11 || 0.20 || 6.80 || x || A || 104-427 || x
+
| 4ANP || 2.11 || 0.20 || 6.80 || 0 || A || 104-427 || x
 
|-
  
|-
−
| 4PAH || 2.00 || 0.17 || 6.80 || x || A || 117-424 || x
 +
| 4PAH || 2.00 || 0.17 || 6.80 || 0 || A || 117-424 || x
 
|-
  
|-
 
| 5PAH || 2.10 || <span style="background:#00FF00">0.16</span> || 6.80 || x || A || 117-424 || x
 
| 5PAH || 2.10 || <span style="background:#00FF00">0.16</span> || 6.80 || x || A || 117-424 || x
 
|-
  
|-
−
| 6PAH || 2.15 || 0.17 || 6.80 || x || A || 117-424 || x
+
| 6PAH || 2.15 || 0.17 || 6.80 || 0 || A || 117-424 || x
 
|}
  
|}
 
<small><center>'''<caption>''' ...</caption></center></small>
  
<small><center>'''<caption>''' ...</caption></center></small>

Revision as of 15:48, 6 August 2013

Page still under construction!!!

Summary

In Task 8 the sequence of PAH was used for finding mutational effects, now the structure will be taken for these analysis. But how to find out, if a mutation changes the structure? Therefore, one calculates the energy of all atoms for the wildtype and the mutated structure and compares the results for changes. There are two different methods for this calculations given: Quantum Mechanics (QM) and Molecular Mechanics (MM). In QM the energy of all electrons in a protein is calculated. It is one of the most accurated methods, but it is very time consuming. In MM the energy of a system is calculated as a function of nuclear positions. It is very fast and easy to calculate, but it ignores electronic motions and is not as accurate as QM. Since QM is too time intensive and the results of MM are nearly as good as the ones calculated with QM, we use MM for the further analysis. Molecular Mechanics uses force fields for the energy calculation, which is defined as a sum of terms. The terms are non-bonded (electrostatic and Van-der-Waals) and bonded (Bond stretching, Angle stretching, bond rotation) interactions. For the structure based mutation analysis the SCWRL and FoldX webserver were used.

Preparation

Lab journal

Structure selection
In some Tasks before, we used the protein structure of 2PAH as reference, but now we have to check some more constraints for the protein structure selection:

  • Structure with the highest resolution (small Å value)
  • Smallest R-factor
  • Highest coverage
  • pH-value ideally near physiological pH of 7.4
  • No gaps (missing residues) included in the structure, so a consecutive numbering of residues should be given

To check which protein structure to use for further analysis, we compared the constraint data for all sequences given in the PAH (P00439) Uniprot entry. <figtable id="pro-struc">

Protein Resolution(Å) R-factor pH Gaps Chain Positions in P00439 Coverage %
1DMW 2.00 0.20 6.80 0 A 118-424 x
1J8T 1.70 0.20 6.80 0 A 103-427 x
1J8U 1.50 0.16 6.80 0 A 103-427 x
1KW0 2.50 0.22 6.80 0 A 103-427 x
1LRM 2.10 0.21 6.80 0 A 103-427 x
1MMK 2.00 0.20 6.80 0 A 103-427 x
1MMT 2.00 0.21 6.80 0 A 103-427 x
1PAH 2.00 0.18 6.80 0 A 117-424 x
1TDW 2.10 0.21 6.80 aa-change: A 117-424 x
1TG2 2.20 0.21 6.80 aa-change: A 117-424 x
2PAH 3.10 0.25 7.00 0 A/B 118-452 x
3PAH 2.00 0.18 6.80 0 A 117-424 x
4ANP 2.11 0.20 6.80 0 A 104-427 x
4PAH 2.00 0.17 6.80 0 A 117-424 x
5PAH 2.10 0.16 6.80 x A 117-424 x
6PAH 2.15 0.17 6.80 0 A 117-424 x
...

</figtable> All proteins were found with the X-ray diffraction method. In <xr id="pro-struc"/> we can see, that the protein 1J8U has a better resolution value as well as R-factor than the other proteins...


Visualisation of used mutations
Following five mutations are mapped to the crystal structure:

Substitution Prediction Database
???Gly103Ser neutral-wrong??? HGMD
Gln172His neutral dbSNP
Ala259Val non-neutral HGMD
Thr266Ala non-neutral dbSNP
Phe392Ser non-neutral dbSNP
Pro416Gln non-neutral HGMD

Mutated structure creation
...

Energy comparisons

...

FoldX

...

Minimise

...

References

<references/>

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