CD task7 protocol
From Bioinformatikpedia
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
#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
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
Gromacs
run repairPDB for original and all scwrl files, example call for original:
/opt/SS12-Practical/scripts/repairPDB 2O4H.pdb -jprot -chain A > 2O4H.repair.pdb
Call Scwrl4 again to make sure all's ok:
/opt/SS12-Practical/scwrl4/Scwrl4 -i 2O4H.repair.pdb -o 2O4H.out.scwrl.pdb
Run pdb2gmx:
/opt/SS12-Practical/gromacs/bin/pdb2gmx -f 2O4H.repair.pdb -o 2O4H.gro -p 2O4H.top
after inputting this, you're asked to choose ForceField and Water Model numbers (we chose 1 and 1)
Run grompp:
/opt/SS12-Practical/gromacs/bin/grompp -v -f grompp.mdp -c 2O4H.gro -p 2O4H.top -o 2O4H.tpr
Minimise system:
/opt/SS12-Practical/gromacs/bin/mdrun -v -deffnm 2O4H.tpr
Analyse minimisation:
g_energy -f 2O4H.edr -o energy_2O4H_1.xvg
(results in gatzmannf/task7/gromacs/Analysis_results.txt)