CD task7 protocol

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)