Structure-based mutation analysis HEXA

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.

• [rs61731240: His -> Asp]

• [rs121907974: Phe -> Ser]

• [rs61747114: Leu -> Phe]

• [rs1054374: Ser -> Ile]

• [rs121907967: Trp -> TER]

• [rs1800430: Asn -> Asp]

• [rs121907982: Ile -> Val]

• [rs121907968: Trp -> Arg]

Protocoll - Using the methods

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

Gromacs:

fetchpdb - done repairPDB - done SCWRL -done pdb2gmx:

        watermodel: TIP3P
       -> done very fast

description of the MDP file: later

Forcefiled 1: AMBER3 Forcefield 2: AMBER99SB-ILDN Forcefiled 3: GROMOS96 53a6 force field (JCC 2004 vol 25 pag 1656)