Difference between revisions of "CD task7 protocol"

From Bioinformatikpedia
Line 27: Line 27:
 
REMARK 465 1 LEU A 312
 
REMARK 465 1 LEU A 312
 
REMARK 465 1 HIS A 313
 
REMARK 465 1 HIS A 313
  +
  +
==Scwrl==
  +
  +
#first nine residues are missing
  +
# read sequence
  +
line = open("./aspa_crop.fasta","r").readlines()[1]
  +
#make lower case
  +
seq = line.lower().strip("\n")
  +
print(line)
  +
  +
#(posi, orig aa, mut aa)
  +
muts = [(285, "e", "a","E285A"),(305,"a","e","A305E"),(123,"g","e","G123E"),(71,"r","h","R71H"),(71,"r","k","R71K"),(213,"k","e","K213E"),(278,"v","m","V278M"),(82,"m","t","M82T"),(235,"e","k","E235K"),(270,"i","t","I270T")]
  +
  +
for mut in muts:
  +
#check if looking at correct residue
  +
if not seq[mut[0]-10] == mut[1]:
  +
print mut, 'wrong residue match'
  +
else:
  +
l = list(seq)
  +
l[mut[0]-10] = mut[2].upper()
  +
newSeq = "".join(l)
  +
st = mut[3] + ".fasta"
  +
outfile = open(st,"w")
  +
# st = "> " + mut[3] + "\n"
  +
# outfile.write(st)
  +
outfile.write(newSeq)
  +
  +
callfile = open("./callSCWRL.sh","w")
  +
  +
for mut in muts:
  +
st = "/opt/SS12-Practical/scwrl4/Scwrl4 -i 2O4H_chainA.pdb -o " + mut[3] + "_scwrl.pdb" + " -s " + mut[3] + ".fasta\n"
  +
callfile.write(st)
  +
  +
/opt/SS12-Practical/scwrl4/Scwrl4 -i 2O4H_chainA.pdb -o E285A_scwrl.pdb -s E285A.fasta
  +
   
 
==FoldX==
 
==FoldX==

Revision as of 20:48, 20 June 2012

Choosing structure

So, we decided to use 2O4H, since it has a bound substrate, low resolution and a listed pH value.

Apo-structure:       2O53 Resolution: 2,7,  R-free: 0,269, pH: 6.0, chains: A,B
Holo-structure:      2O4H Resolution: 2,7,  R-free: 0,271, pH: 6.0, chains: A,B intermediate substrate analog: N-phosphonomethyl-L-aspartate
Apo-structure:       2I3C Resolution: 2,8,  R-free: 0,243, pH: -  , chains: A,B
Ensemble Refinement  2Q51 Resolution: 2,8,  R-free: 0,239, pH: -  , chains: A,B

For 2O4H, there are only missing residues at the N- and C-terminal ends, which can be neglected:

REMARK 465 MISSING RESIDUES                                                     
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE                       
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN               
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.) 
REMARK 465   M RES C SSSEQI                                                     
REMARK 465   1 ALA A    -1                                                      
REMARK 465   1 ILE A     0                                                      
REMARK 465   1 ALA A     1                                                      
REMARK 465   1 THR A     2                                                      
REMARK 465   1 SER A     3                                                      
REMARK 465   1 CYS A     4                                                      
REMARK 465   1 HIS A     5                                                      
REMARK 465   1 ILE A     6                                                      
REMARK 465   1 ALA A     7                                                      
REMARK 465   1 GLU A     8                                                      
REMARK 465   1 CYS A   311                                                      
REMARK 465   1 LEU A   312                                                      
REMARK 465   1 HIS A   313

Scwrl

  1. first nine residues are missing
  2. read sequence

line = open("./aspa_crop.fasta","r").readlines()[1]

  1. make lower case

seq = line.lower().strip("\n") print(line)

  1. (posi, orig aa, mut aa)

muts = [(285, "e", "a","E285A"),(305,"a","e","A305E"),(123,"g","e","G123E"),(71,"r","h","R71H"),(71,"r","k","R71K"),(213,"k","e","K213E"),(278,"v","m","V278M"),(82,"m","t","M82T"),(235,"e","k","E235K"),(270,"i","t","I270T")]

for mut in muts:

       #check if looking at correct residue
       if not seq[mut[0]-10] == mut[1]:
               print mut, 'wrong residue match'
       else:
               l = list(seq)
               l[mut[0]-10] = mut[2].upper()
               newSeq = "".join(l)
               st = mut[3] + ".fasta"
               outfile = open(st,"w")
  1. st = "> " + mut[3] + "\n"
  2. outfile.write(st)
               outfile.write(newSeq)

callfile = open("./callSCWRL.sh","w")

for mut in muts:

       st = "/opt/SS12-Practical/scwrl4/Scwrl4 -i 2O4H_chainA.pdb -o " + mut[3] + "_scwrl.pdb" + " -s " + mut[3] + ".fasta\n"
       callfile.write(st)

/opt/SS12-Practical/scwrl4/Scwrl4 -i 2O4H_chainA.pdb -o E285A_scwrl.pdb -s E285A.fasta


FoldX

list.txt

2O4H.pdb

individual_list.txt

EA285A;
AA305E;
GA123E;
RA71H;
RA71K; 
KA213E;
VA278M;
MA82T;
EA235K;
IA270T;

run.txt

<TITLE>FOLDX_runscript;
<JOBSTART>#;
<PDBS>#;
<BATCH>list.txt;
<COMMANDS>FOLDX_commandfile;
<BuildModel>#,individual_list.txt;
<END>#;
<OPTIONS>FOLDX_optionfile;
<Temperature>298;
<R>#;
<pH>7;
<IonStrength>0.050;
<water>-CRYSTAL;
<metal>-CRYSTAL;
<VdWDesign>2;
<OutPDB>true;
<pdb_hydrogens>false;
<complex_with_DNA> true; 
<END>#;
<JOBEND>#;
<ENDFILE>#;

command

/opt/SS12-Practical/foldx/FoldX.linux64 => choose 3 => run.txt

Minimise

We prepared the .pdb files with pymol (remove hydrogens, solvent)

/opt/SS12-Practical/minimise/minimise E285A_scwrl_repair.pdb E285A_scwrl_min.pdb