Difference between revisions of "Structure-based mutation analysis HEXA"

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(Gromacs)
(Results)
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== Results ==
 
== Results ==
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=== Energy ===
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==== FoldX ====
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{| border="1" style="text-align:center; border-spacing:0;"
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|Mutation
  +
|energy value
  +
|Ratio difference
  +
|-
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|wildtype
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| -154.17
  +
|1.56
  +
|-
  +
|Rs61731240
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| -151.61
  +
|0.16
  +
|-
  +
|Rs121907974
  +
| to do
  +
|todo
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|-
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|Rs61747114
  +
| -153.78
  +
|0.15
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|-
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|Rs1054374
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| -152.15
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|1.21
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|-
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|Rs121907967
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| -
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| -
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|-
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|Rs1800430
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| -155.11
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| -0.72
  +
|-
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|Rs121907982
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| -154.36
  +
| -0.23
  +
|-
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|Rs121907968
  +
| -149.08
  +
|3.20
  +
|-
  +
|}

Revision as of 18:23, 10 August 2011

Farbcode bei active side: active side: grün Glycolysation: gelb Cystein: cyan Mutation:rot

Sequence Description

We had to use a PDB file, in which are no missing residues and the quality of the structure should be high. We found only one PDB structure which was not bounded to a ligand. Therefore, we could not regard the quality and the pH value, the R-factor and the coverage. Nevertheless, we listed in the following table this values:

experiment type X-Ray diffraction
Resolution 2.8 Å
temperature (Kelvin) 100K
temperature (Celsius) -173 °C
pH-Value 5.5 (slightly acid)
R-Value 0.270


It was not possible to find one file, without any missing residues. In each file there was a gap between residue 74 to 89 and the last amino acid. Therefore, we decided to cut off the first 89 residues and use a PDB file with a structure from 89 - 528. This file can be found [here].

Mutations

Because of the shorten PDB file, it was not possible for us to analyse the first two mutations on position 29 and 39.

SNP-id codon number mutation codon mutation triplet
rs4777505 29 Asn -> Ser AAC -> AGC
rs121907979 39 Leu -> Arg CTT -> CGT
rs61731240 179 His -> Asp CAT -> GAT
rs121907974 211 Phe -> Ser TTC -> TCC
rs61747114 248 Leu -> Phe CTT -> TTT
rs1054374 293 Ser -> Ile AGT -> ATT
rs121907967 329 Trp -> TER TGG -> TAG
rs1800430 399 Asn -> Asp AAC -> GAC
rs121907982 436 Ile -> Val ATA -> GTA
rs121907968 485 Trp -> Arg gTGG -> CGG

Analysis of the mutations

We created for each mutation an extra page. The summary of the analysis can be seen in the Summary Section.


Protocoll - Using the methods

Pymol

We visualized the local hydrogen-bonding network with following commands:

distance hbonds, all, all, 3.2, mode=2
zoom resi <interval>
hide labels, all
color red, resi <mutation_position>

Furthermore, we also used the polar contact mode in pymol to visulize the h-bonds.

The clashed are visualized by the following commands:

distance clash = pos_mutation, all, 2.0, 0
zoom clash

FoldX

To use FoldX, we created a runfile, which can be found [here]. We fitted the temperature and pH-value to the values we extracted from the PDB page. Furthermore, we analysed the mutations with a random choosen temperature and pH value, to see how much influence these parameteres have on the analysis.

We ran FoldX with following command:

FoldX -runfile run.txt > foldx_output


minimise

Next we used minimise. Therefore, it was not necessary to create any file for the run. Sadly, we could not find any documentation about minimise and therefore, it was really hard to figure out how it works and what means the output.

We ran minimise with following command:

minimise <input> <output>

Gromacs

Before we could run Gromacs, we had to curate our PDB file. Therefore, we used the script repairPDB to extract chainA. Next we run SCRWL to make sure, that every residue is available in the PDB file.

Then we used the commands which are listed in our task section.


Additionally, to the analysis of our mutated sequences, we also chose different forcefields and analysed our protein without any mutation with these forcefields. Here are the results of this analysis:

analysed energies (in kJ/mol) force field
AMBER3 AMBER99SB-ILDN CHARMM27
Bond Average 852.968 1091.57 1796.6
Err Estimation - 270 240
RMSD 42.0241 -nan 2924.29
Drift -74.0853 -1622.75 -1404.11
Angle Average 3438.47 3326.81 4764.7
Error Estimation - 62 60
RMSD 16.8864 -nan 466.82
Drift -33.7041 404.076 368.45
Potential Average -50917.7 -61304.1 166.582
Error Estimation - 960 39
RMSD 66.4149 -nan 79.7058
Drift -132.636 -6402.44 280.841

Furthermore, we used different numbers for nsteps. The result of how long these analysis run, can be found in the following table and graph:

nstep time real time user time sys #steps
50 8.268s 3.860s 0.110s 24
500 27.523s 47.650s 0.540s 321
5000 25.281s 17.710s 0.210s 114
50000 14.940s 14.210s 0.300s 91

Results

Energy

FoldX

Mutation energy value Ratio difference
wildtype -154.17 1.56
Rs61731240 -151.61 0.16
Rs121907974 to do todo
Rs61747114 -153.78 0.15
Rs1054374 -152.15 1.21
Rs121907967 - -
Rs1800430 -155.11 -0.72
Rs121907982 -154.36 -0.23
Rs121907968 -149.08 3.20