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

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

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