Lab Journal Hemochromatosis Task9

Structure Selection

For the calculation of the coverage, the signal peptide at the beginning was omitted:

coverage = ( 275 (length of sequence used in crystallisation ) - 3 ( missing residues ) ) / ( 348 ( length of Q30201 ) - 22 ( length of signal peptide ) )

3D mutations with SCWRL4

First, the wild type sequence was extracted from the FASTA entry of 1A6Z on rcsb.org. Then, using mut_seq.py (see below for the code), all mutant sequences were generated. The mutated structures were then generated with SCWRL4 using the following command

Scwrl4 -i <1a6z chain A pdb file> -o <mutated pdb> -s <mutated sequence file>

To check, whether only the right residue was mutated, the sequence of the PDB files containing the mutations were extracted and compared. The mutations are colored in red in the following:

RSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTYQVEHPGLDQPLIVIW
RSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDDESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIW
RSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHIFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIW
RSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVISSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIW
RSHSLHYLFMGASEQDLGLSLFEALGYMDDQLFVFYDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQVILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNRAYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWLKDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIW

3D mutations with FoldX

FoldX was run with modified scripts from the tutorial page of FoldX.

Minimisation

The minimisation was carried out on the student machines with the following command

/opt/SS12-Practical/minimise/minimise <inPDB> <outPDB> > <logfile>

1 kJ = 0.239 kcal

mut_seq.py

<source lang="python"> import copy seq="rllrshslhylfmgaseqdlglslfealgyvddqlfvfydhesrrveprtpwvssrissqmwlqlsqslkgwdhmftvdfwtimenhnhskeshtlqvilgcemqednstegywkygydgqdhlefcpdtldwraaeprawpt klewerhkirarqnraylerdcpaqlqqllelgrgvldqqvpplvkvthhvtssvttlrcralnyypqnitmkwlkdkqpmdakefepkdvlpngdgtyqgwitlavppgeeqrytcqvehpgldqpliviw" seq= list(seq)

mut = {53:"M",63:"D",97:"I",217:"I",282:"Y"}

for key in mut.iterkeys(): mut_seq = copy.deepcopy(seq) mut_seq[key-23] = mut[key] ## -(22+1) 22 for PDB - seq offset and 1 for indexing starting at 0 fName= str(key) + mut[key] + ".seq" with open(fName,"w+") as f: f.write("".join(mut_seq[3:])) #leave out first three residues because they are missing in the PDB structure

</source>

The minimisation of all structures was executed with the following script:

min_structures.sh

<source lang="bash">

fx=./foldx/*.pdb

for f in $fx do base=${f%.*} mkdir $base

/opt/SS12-Practical/minimise/minimise $f $base/iter1.pdb > $base/iter1.out

for i in 2 3 4 5 do p=$(($i-1)) /opt/SS12-Practical/minimise/minimise $base/iter$p.pdb $base/iter$i.pdb > $base/iter$i.out done

done </source>

The results were extracted with

get_res.py

<source lang="python"> import sys import os from collections import defaultdict

folder = sys.argv[1]

mutNames = os.listdir(folder) eList = defaultdict(list) for mut in sorted(mutNames): if not mut.endswith(".pdb"): continue mFolder = folder + "/" + mut[:-4] for i in range(1,6): fName = mFolder + "/iter"+ str(i) + ".out" with open(fName) as f: t = f.readlines() energy = t[-6].strip()[7:].strip(")") energy = str(round(float(energy),2)) eList[mut[:-4]].append(energy)

for mut in sorted(eList.keys()): eList[mut].insert(0,mut) print "| " + " || ".join(eList[mut]) print "|-"

</source>