6.

Fix the given protein structure to remove anisotropic thermal parameters, alternate conformers, non
standard or incomplete residues (1).
 
grep -Ei "^atom|^end|^hetatm" model.pdb | awk '{print $4}'| sort | uniq
 
A
AASP
AGLU
ALA
ARG
ASN
ASP
BASP
BGLU
CYS
GLN
GLU
GLY
HIS
ILE
LEU
LYS
MET
MSE
PHE
PRO
SER
THR
TRP
TYR
VAL
 
Ran the above mentioned command to check for non standard amino acids. Used Pymol to mutate the
MSE to the parent amino acid Methionine.
 
PyMOL>sele resn 'MSE'
Using Wizard Mutagenesis the MSE was mutated to Methionine. Saved file using:
 
save clean_noMSE.pdb
Cleaned the file to get rid of anisou and alternate conformers using the following command.
 
awk '((substr($0,17,1)=="A" || substr($0,17,1)==" ")) {print substr($0,1,16), substr($0,18,63)} '
clean_noMSE.pdb|  grep -Ei "atom|end|ter" > cleaned.pdb
 
To get rid of any incomplete residues Discovery Studio was used.
Edit --> Preferences --> Protein utilities --> Clean Protein
 
 
 
 
 
 
 
Saved as cleaned.pdb.
 
7. Do 2000 steps of Steepest Descent minimization with an emtol value of 10 kJ/mol cut off or until
convergence under vacuum conditions. Report the change in (i) potential energy and (ii) short range
electrostatic interaction energy between step 0 and the last step of miniization. (2)
 
Command 1:
gmx pdb2gmx -f cleaned.pdb -o processed.gro -water none -ignh
 
Number of bonds was 5223, now 5223
Generating angles, dihedrals and pairs...
Before cleaning: 13792 pairs
Before cleaning: 13897 dihedrals
Keeping all generated dihedrals
Making cmap torsions...
There are 13897 dihedrals, 1043 impropers, 9449 angles
          13660 pairs,     5223 bonds and     0 virtual sites
Total mass 37070.960 a.m.u.
Total charge -22.000 e
 
Downloaded minim.mdp and changed emtol to 10 and nsteps to 2000.
 
Command 2: gmx grompp -f minim.mdp -c processed.gro -p topol.top -o em.tpr -maxwarn 2
Command 3: gmx mdrun -v -deffnm em
 
Steepest Descents did not converge to Fmax < 10 in 2001 steps.
Potential Energy  = -5.6005031e+04
Maximum force     =  8.8561835e+02 on atom 4753
Norm of force     =  1.9960043e+01
 
 
 
 
Step 0:
 Energies (kJ/mol)
           Bond          Angle    Proper Dih. Ryckaert-Bell.          LJ-14
    2.59933e+04    3.72670e+03    1.16665e+02    5.41337e+03    1.38584e+04
     Coulomb-14        LJ (SR)   Coulomb (SR)   Coul. recip.      Potential
    4.25853e+04    2.73387e+04   -1.03494e+05    1.32584e+04    2.87965e+04
 Pressure (bar)
    4.00791e+04
 
Step           Time
           2000     2000.00000
 
   Energies (kJ/mol)
           Bond          Angle    Proper Dih. Ryckaert-Bell.          LJ-14
    7.34532e+02    2.98045e+03    1.79105e+02    3.94844e+03    6.41973e+03
     Coulomb-14        LJ (SR)   Coulomb (SR)   Coul. recip.      Potential
    4.24319e+04   -9.90076e+03   -1.10273e+05    7.47492e+03   -5.60050e+04
 Pressure (bar)
   -3.87206e+02
 
8. Compare the starting structure and final minimized structure. Report RMSD for i) all atoms, ii) main
chain atoms and iii) C-alpha atoms. (you can use any tool of your choice). (2)
 
Command: gmx trjconv -f em.gro -o em.pdb -pbc mol -s em.tpr (Selected 1)
 
RMSD reported using LSQMAN: (Commands highlighted in Red)
All: The   2594 atoms have an RMS distance of    0.604 A
CA: The    327 atoms have an RMS distance of    0.358 A
Main Chain: The    981 atoms have an RMS distance of    0.377 A
New directory made:
9. Soak the given
molecule (do not use the
minimized one, unless
you fixed pbc errors!) in a
box of water molecules,
covering at least 8 Å from
the surface of the
molecule in all directions.
Add proper ions to
neutralize the system.
Have 150 mM of NaCl in
the simulation system. (1)
 
Command: gmx pdb2gmx
-f cleaned.pdb -o
processed.gro -water spce
-ignh
 
Number of bonds was
5223, now 5223
Generating angles,
dihedrals and pairs...
Before cleaning: 13792 pairs
Before cleaning: 13897 dihedrals
Keeping all generated dihedrals
Making cmap torsions...
There are 13897 dihedrals, 1043 impropers, 9449 angles
          13660 pairs,     5223 bonds and     0 virtual sites
Total mass 37070.960 a.m.u.
Total charge -22.000 e
 
Create the box
Command: gmx editconf -f processed.gro -o newbox.gro -c -d 0.8 -bt cubic
  
Fill it
with

solvent:
Command: gmx solvate -cp newbox.gro -cs spc216.gro -o solv.gro -p topol.top
 
Generate ions.tpr
Command: gmx grompp -f ions.mdp -c solv.gro -p topol.top -o ions.tpr
 
Neutralize with ions:
Command: genion -s ions.tpr -o solv_ions.gro -p topol.top -pname NA -nname CL -neutral -conc
0.15
 
10. Equilibrate the added water molecules and ions by energy minimization (SD 1000 steps - emtol
10kJ/mol followed by CG 1000 steps -emtol 1kJ/mol), NVT and NPT runs for 10ps each, followed by
100 ps unrestrained MD simulation. Write the coordinates and energy parameters at every 100 fs (2)
 
Changed the minim.mdp to Integrator=steep, emtol=10 and nsteps= 20000 (instead of 2000 as it gives a
problem for CG later)
 
Generated the em-sd.tpr
Command: gmx grompp -f minim.mdp -c solv_ions.gro -p topol.top -o em-sd.tpr
 
Minimization:
Command: gmx mdrun -v -deffnm em-sd
 
Steepest Descents converged to machine precision in 11217 steps,
but did not reach the requested Fmax < 10.
Potential Energy  = -1.1578285e+06
Maximum force     =  3.6352853e+02 on atom 4753
Norm of force     =  4.1390568e+00
 
Changed minim.mdp into Integrator=cg, emtol=1 and nsteps=1000.
 
Generated the em-sd.tpr
Command: gmx grompp -f minim.mdp -c em-sd.gro -p topol.top -o em-cg.tpr
 
Minimization:
Command: gmx mdrun -v -deffnm em-cg
 
Energy minimization reached the maximum number of steps before the forces
reached the requested precision Fmax < 1.
 
writing lowest energy coordinates.
 
Polak-Ribiere Conjugate Gradients did not converge to Fmax < 1 in 1001 steps.
Potential Energy  = -1.1659402e+06
Maximum force     =  5.4668518e+02 on atom 282
Norm of force     =  6.4200106e+00
 
nvt.mdp was editied for 10ps and generating log every 500fs.
; Run parameters
integrator              = md       
nsteps                  = 5000    
dt                      = 0.002    
; Output control
nstxout                 = 250      
nstvout                 = 250      
nstenergy               = 250     
nstlog                  = 250      
 
Generate the nvt.tpr file:
Command: gmx grompp -f nvt.mdp -c em-cg.gro -r em-cg.gro -p topol.top -o nvt.tpr -maxwarn 1
 
MD run:
Command: gmx mdrun -v -deffnm nvt
 
npt.mdp was editied for 10ps and generating log every 500fs.
; Run parameters
integrator              = md       
nsteps                  = 5000    
dt                      = 0.002    
; Output control
nstxout                 = 250      
nstvout                 = 250      
nstenergy               = 250     
nstlog                  = 250      
 
Generate the npt.tpr file:
Command: gmx grompp -f npt.mdp -c nvt.gro -r nvt.gro -p topol.top -o npt.tpr -maxwarn 1
 
MD run:
Command: gmx mdrun -v -deffnm npt
 
 
md.mdp was editied for 100ps and generating log every 500fs.
 
; Run parameters
integrator              = md      
nsteps                  = 50000   
dt                      = 0.002    
; Output control
nstxout                 = 0        
nstvout                 = 0        
nstfout                 = 0        
nstenergy               = 250     
nstlog                  = 250     
nstxout-compressed      = 250     
 
Generate the md tpr file:
Command: gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -o md_0_1.tpr -maxwarn 1
 
MD run:
Command: gmx mdrun -v -deffnm md_0_1
 
 
 
11. Plot the rmsd of the trajectory comparing against the starting conformer. (2).
 
Commands:
 
gmx trjconv -s md_0_1.tpr -f md_0_1.xtc -o md_0_1_noPBC.xtc -pbc mol -center (Choose 1 and 0)
 
Initital Structure: gmx rms -s md_0_1.tpr -f md_0_1_noPBC.xtc -o rmsd.xvg -tu ns (Choose 4 and 4)
 
Structure post MD: gmx rms -s em.tpr -f md_0_1_noPBC.xtc -o rmsd_xtal.xvg -tu ns (Choose 4 and
4)
 
Plot the two together using Xmgrace: xmgrace rmsd_xtal.xvg  rmsd.xvg
 
 
Black = New Structure; Red =Old Structure