gmx pdb2gmx -f clean-coot.pdb -o protein.

gro -water none -ignh

gmx editconf -f protein.gro -o box.gro -c -d 1.0 -bt cubic
gmx grompp -f em_steep.mdp -c protein.gro -p topol.top -o em_steep.tpr
gmx mdrun -v -deffnm em_steep
gmx trjconv -f em_steep.gro -o em_steep_vac.pdb -pbc mol -s em_steep.tpr
(to save minimised structure as PDB)
This is to prevent molecule from breaking. Group 1 was selected to choose
protein and H.

For RMSD comparison on lsqman

re m1 clean-coot.pdb
re m2 em_steep_vac.pdb
ex m1 A1-3000 m2 A1
at ca (default)
at all
at ma

gmx pdb2gmx -f clean-coot.pdb -o protein.gro -water spc -ignh

gmx editconf -f protein.gro -o box.gro -c -d 0.9 -bt cubic
gmx solvate -cp box.gro -cs spc216.gro -o solv.gro -p topol.top
gmx grompp -f em_steep.mdp -c solv.gro -p topol.top -o ions.tpr
gmx genion -s ions.tpr -o ions.gro -p topol.top -pname NA -nname CL -conc
0.15 -neutral
gmx grompp -f em_steep.mdp -c ions.gro -p topol.top -o em_steep.tpr
gmx mdrun -v -deffnm em_steep
gmx grompp -f em_cg.mdp -c em_steep.gro -p topol.top -o em_cg.tpr
gmx mdrun -v -deffnm em_cg -nt 3
gmx grompp -f nvt.mdp -c em_cg.gro -r em_cg.gro -p topol.top -o nvt.tpr -
maxwarn 3
gmx mdrun -v -deffnm nvt -nt 3
gmx grompp -f npt.mdp -c nvt.gro -r nvt.gro -p topol.top -o npt.tpr -
maxwarn 3
gmx mdrun -v -deffnms npt -nt 3
gmx grompp -f md.mdp -c npt.gro -p topol.top -o md.tpr -maxwarn 3
gmx mdrun -v -deffnm md -nt 3
gmx trjconv -s md.tpr -f md.xtc -o md_process.xtc -pbc mol -center
(Choose 1 and 0)
gmx energy -s md.tpr -f md.edr -o srwv.xvg (for short range van der Waal
interaction plot)
gmx rms -s md.tpr -f md_process.xtc -o rmsd.xvg (for RMSD plot)
gmx rms -s em_cg.tpr -f md_processed.xtc -o og_rmsd.xvg (wrt original
structure)
xmgrace srwv.xvg
xmgrace rmsd.xvg
gmx rmsf -s md.tpr -f md_processed.xtc -o rmsf.xvg -oq bfac.pdb
On xmgrace, data-> transformation -> running average (set to 10)

awk '$10 > 50' bfac.pdb |less

Identifying potential ligand binding sites:
Opened the receptor molecule in discovery studio.
Chemistry explorer -> Hydrogen -> Add Polar

Click on receptor-ligand interactions: -> From receptor cavities -> The

binding sites are shown in the side panel along with a representation on
the receptor molecule of the binding site 1

15 Potential binding sites were identified on the receptor molecule.

Docking:

adt; opens AutoDockTools

Opened both receptor and ligand molecule.
File -> Read Molecule
Generating pdbqt files of both receptor and ligand:
Ligand:
Ligand(taskbar above the view window) -> input -> choose ligand
Ligand(taskbar above the view window) -> output -> save as PDBQT
Receptor:
Grid -> Macromolecule -> choose -> receptor.pdb -> saved as PDBQT

Open the pdbqt files of both the receptor and ligand.

Running Autogrid:
Grid -> Grid Box -> adjust parameters to cover the whole molecule in the
box

Grid -> output -> .gpf file is saved

Run -> autogrid -> give log file name with .glg extension and include the
log filename in the command and launch.
Autogrid log file is generated.

Running autodock:
1)Docking > Macromolecule > Set Rigid filename> Select the receptor pdbqt
> Open
2-Docking > Ligand > Choose> Click ‘Ligand’ > Select Ligand.pdbqt >
Accept
3-Docking > Search Parameters > Genetic Algorithm> Accept
4-Docking > Output > Lamarckian GA> Save file as ‘.dpf’
5-Run > Run AutoDock

Observe the energy of each binding from the log file.

grep "Estimated Free Energy of Binding" receptor1.dlg

Analysis:
1-Analyze > Docking > Open> Select ‘dock.dlg’ > Open >Assign Ligand New
Name > OK
2-Analyze > Macromolecule > Choose> Click ‘Protein’ > Select
Macromolecule
3- Analyze > Conformations> Play, Ranked By Energy

lig2
VINA
obabel -ipdb lig1.pdb -opdbqt -O lig1.pdbqt –partialcharges gasteiger -p
7.0
vina --config config.txt --log lig2_vina.log --ligand lig2.pdbqt
(#the config.txt file is filled with the box parameters centred around
the ligand in it’s best binding according to the blind docking done in
autodock. Vina gives the best ligand conformation. The output is a .pdbqt
file)

LIG1

OUTPUT:

LIG2:

OUTPUT

Lig3:

OUTPUT

CONVERTING THE OUTPUT FILES(.pdbqt) TO PDB files

obabel -ipdbqt lig1_out.pdbqt -opdb -O lig1_out.pdb

The output pdb file contains all the 9 conformations.

The modest showing least energy(best conformation) was extracted to a new

PDB file. This PDB File is further used to view the interactions.

2D RECEPTOR LIGAND INTERACTIONS:

This is visualized in discovery studio:

The receptor molecule was loaded.
Onto the same window the pdb file of the best conformation of the ligand
was loaded:
File -> insert from -> file -> Ligand file
The ligand appears on the binding site.
In the side window, below Receptor ligand interactions; 2D interactions
is given as a option.

It is clicked to see the interactions as a 2D diagram.

CHEMICAL MODIFICATIONS FOR THE LIGAND

The molecule with best conformation was loaded along with the receptor on
DS

3D interaction of the molecule were observed. When checked for H-bonds,

sites with acceptor regions but no donors in the ligand were observed.

An H-Bond Donating Group was added to the identified site using coot.
The SMILEs string of ligand 2 was generated from DS.
This was loaded as a 2D structure in COOT.
Ligand -> SMILES to 2D -> give the SMILES string in the box
Necessary chemical modifications were performed using the toolbox.
The SMILE string of modified ligand was saved from the smile icon in the
Ligand Builder window.

The new SMILES string was loaded in DS and saved as a PDB file.

Docking was performed again using VINA.:

The PDB file was converted to pdbqt file using obabel:
obabel -ipdb lig5.pdb -opdbqt -O lig5.pdbqt
VINA was run using the command as before.
Increased affinity was observed across all conformations.

Hence adding the group was successful in increasing the binding affinity.