DOCKING#MOLECULARSIMULATION#ENERGYMINIMISATION

STRUCTURAL BIOINFORMATICS PRACTICAL-INTERNAL (03/10/2023, FRIDAY) 1
STRUCTURAL BIOINFORMATICS PRACTICAL-
INTERNAL (03/10/2023, FRIDAY)
NAME: Sahastranshu Pandey ROLL NO.: 225HSBB030
SECTION-1: MOLECULAR EDITING
Q1. (i) The SMILES string that we got is: COc1cc(ccc1)-c(o2)c(C@@HC(O)O)c(c23)ccc(c3)-c(ccc4)cc4OC
(ii) We will get its molecular structure with the help of Discovery Studio by: File→ New→ Molecular Window→ File→ Insert From→
SMILES.
(iii) The structure that we got is:

(iv) Now, we will save the coordinates in PDB format by: File→ Save As → Files of Type: PDB(.pdb)
(v) The file is saved in the Current Directory named “ligand1.pdb”.
Q2. (i) We will generate the second structure (ligand2.pdb) with the help of Discovery Studio. We will first check the Chirality by doing:
Structure→ Monitor→ Chirality. We have only one chiral center which is shown below:

(ii) Now, we will change the Chirality by: Select the Chiral Centre→ Chemistry→ Stereochemistry→S. After changing the chirality, the
molecule will look like this:

(iii) Now, we will save this molecule as “Ligand2.pdb” using: File→ Save As→ File Type: PDB(.pdb)
Q3. (i) I got the 2D structure of “ligand1.pdb” in Discovery Studio by: Structure→ Show 2D Structure. The structure looks like this:
I had to change the molecule mentioned above to:

(ii) For changing, I just changed from the N-atom to the H-atom for which I did: Chemistry→ Change Element→ H. After I did this, the
molecule looked like the following:

(iii) I saved it as “ligand3.pdb” by:File→ Save As→ PDB:(.pdb)
Q4. (i) For changing torsion for decreasing distance, I did in Dioscovery Studio:
First select two farthest atoms by selecting two atoms using shift
structure → monitor → Distance (visualize the distance)
Then small molecules→ torsion → then select a bond → rotate using mouse and minimize the distance
Q5. For deleting non-terminal carbon to minimize a structure, I did in Discovery Studio:
Select a non-ternminal carbon
Delete it

Select two non-bonded adjacent atoms to make a bond
Chemistry → bond → single
Clean geometry
SECTION-2:MOLECULAR SIMULATIONS
Q6: Fix the given protein structure to remove anisotropic thermal parameters, alternate conformers, and non-standard or incomplete residues.
(i) For fixing the protein structure, we will use ccp4i in which we will remove the H atoms, anisotropic thermal parameters, and alternate
conformers to get model_noH_noAlt_noAniso.pdb
(ii) After this, we will open this file in PyMol in which we will mutate the MSE residue present at the 56th position to MET (Methionine) with
the help of the following steps:
Terminal→PyMol→GUI opens up.
File→Open→model_cleaned.pdb
Click on Selecting Residues →Click on S
Select MSE from the sequence → A separate object called sele is created
Click on Wizard→ Mutagenesis → Select the same residue → No mutation → Met → Apply →Done
Save this molecule as model_cleaned.pdb using File → Save Molecule → choose state as all →OK → Save
Q7. 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 minimization.
Ans.:
(i) Firstly, opening the em_invacuo.mdp file and changing the following parameters in it:

emtol = 10.0; Stop minimization when the energy changes by less than emtol kJ/mol.
nsteps = 2000; Maximum number of (minimization) steps to perform
(ii) Now, running the following commands:
gmx pdb2gmx -f model_cleaned.pdb -water none -ignh
# Choosing 6
# This command generated a topol.top file, posre.itp file and a conf.gro file
gmx grompp -f em_invacuo.mdp -c conf.gro -p topol.top -o em-steep.tpr
gmx mdrun -v -deffnm em-steep
(iii) Now, we open the em-steep.log file in a text editor. The results that I got are:
Energy Step 1 Step 1999
Potential Energy (Marked as ‘Potential’ in em-steep.log file) 5.79263e+03 1.88995e+04
Short-Range Electrostatic Interaction Energy (Marked as
‘Coulomb (SR)’ in em-steep.log file)
6.01867e+04 6.30853e+04
Q8. 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).
Ans.: (i) I ran the following command to get the ‘em-steep.pdb’ file for comparison:
gmx trjconv -f em-steep.gro -s em-steep.tpr -o em-steep.pdb -pbc mol
# Selecting 0

(ii) Now, comparing the two proteins with the help of LSQMAN with the help of the following commands:
re m1 model_cleaned.pdb
re m2 em-steep.pdb
at all
ex m1 A1-188 m2 A1
#Compares the 2 proteins at all atoms
at ma
ex m1 A1-188 m2 A1
#Compares all main chain ( C-alpha, N and C) atoms

at ca
ex m1 A1-188 m2 A1
#Compares at C-alpha atoms

Q9. Soak the given molecule (do not use the minimized one, unless you fixed pbc errors!) in a box of water molecules, covering at least 9 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.
Ans.: I ran the following commands:
gmx pdb2gmx -f model_cleaned.pdb -water spc -ignh
# Selected 6
gmx editconf -f conf.gro -bt cubic -d 0.9 -o box.gro
gmx solvate -cp box.gro -cs spc216.gro -p topol.top -o solvated.gro
gmx editconf -f solvated.gro -o solvated.pdb
gmx grompp -f em.mdp -c solvated.gro -p topol.top -maxwarn 5 -o forions.tpr
gmx genion -s forions.tpr -p topol.top -o ion.gro -neutral -conc 0.15
# Selected 13
Q10. 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
Ans.: (i) The ‘em.mdp’ file was modified as per the parameters given in the question. the parameters are as follows:

(ii) A copy of the ‘em.mdp’ file named ‘em2.mdp’ was created and modified for running CG energy minimization. The parameters are as
follows:
Parameters of ‘em.mdp’ file
Parameters of ‘em2.mdp’ file

(iii) Then, I ran the following commands:
gmx grompp -f em.mdp -c ion.gro -p topol.top -o ex-steep.tpr
gmx mdrun -v -deffnm ex-steep
gmx trjconv -s ex-steep.tpr -f ex-steep.gro -o ex-steep.pdb -pbc mol
#Select 0
gmx grompp -f em2.mdp -c ex-steep.gro -p topol.top -o ex-cg.tpr
gmx mdrun -v -deffnm ex-cg
gmx trjconv -s ex-cg.tpr -f ex-cg.gro -o ex-cg.pdb -pbc mol
#Select 0
(iv) Now, I made some changes in the nvt.mdp, npt.mdp, and run.mdp which are as follows:
Changes in npt.mdp file

Changes in nvt.mdp file

(v) Now, I ran the following commands:
gmx grompp -f nvt.mdp -c ex-cg.gro -p topol.top -r ex-cg.gro -maxwarn 5 -o nvt.tpr
gmx mdrun -v -deffnm nvt
gmx grompp -f npt.mdp -c nvt.gro -p topol.top -r nvt.gro -maxwarn 5 -o npt.tpr
Changes in run.mdp file

gmx mdrun -v -deffnm npt
gmx grompp -f run.mdp -c npt.gro -p topol.top -r npt.gro -maxwarn 5 -o run.tpr
gmx mdrun -v -deffnm run
Q11. Plot the rmsd of the trajectory comparing against the starting conformer.
Ans.: (i) I ran the following commands:
gmx trjconv -s run.tpr -f run.xtc -o run-fix.xtc -pbc mol
#Select 1
gmx rms -f run-fix.xtc -s run.tpr -o rmsd.xvg
#Select 2(Protein -H) both times
xmgrace rmsd.xvg
(ii) Now, in Xmgrace, I did Data→ Transformation→ Running Average→ Length=10→ Accept

→ For the RMSF graph, I ran the following commands:
gmx rmsf -f run-fix.xtc -res -s run.tpr -oq bfac.pdb -o rmsf.xvg
# Selected 2
xmgrace rmsf.xvg

→ For the Radius of Gyration, I ran the following commands:
gmx gyrate -f run-fix.xtc -s run.tpr -o radius.xvg
# Selected 2
xmgrace radius.xvg

SECTION-3: HOMOLOGY MODELLING
12. (i) We will first go to the site (http://cib.cf.ocha.ac.jp/bitool/MIX/) in which we will copy and paste our query sequence and then we will
select all the prediction methods and then we will click “start prediction”
(ii) After predicting the Secondary Structure of the given sequence from the above-mentioned suite, we got the following result:
(iii) We will also, save the result in the “Prediction_Result.pdf” file by printing the whole web page.
(iv) From this result, we can say that the secondary structure of the given sequence consists of 7 alpha helices and 3 Beta strands.
13. (i) Now we will go blastp to get hits for our “query.fasta” sequence. In Organism, we will select “Klebsiella (taxid:570)” and then we will hit
BLAST.
(ii) After doing this, we got the following result:
Query Coverage: 91%
Percent Identity: 90.53%
PDB code: 2MBS
Chain ID: 2MBS_A

(iii) Now, we will download the file by going to the RCSB PDB page → Typing “2MBS” in the search box→ Download Files→ PDB format.
The file will be downloaded as “2mbs.pdb”.
14. (i) Now, we will clean the file with the help of ccp4i to get the “2mbs_cleaned.pdb” file.
(ii) Now, we will open it in Chimera and save the sequence with the help of : Tools→ Sequence→ Sequence→ File→ Save As→2mbs.fasta
file.

(iii) Now, we will open ClustalW and will give query2 sequence and 2mbs.fasta sequence as an input and then will obtain the “clustalw.pir” file.
Now, we will edit the file according to the Modeller Alignment Format as shown below:
15. (i) Now we will run the following command to generate 3 models:
mod10.4 model-default.py
(ii) The three models that were generated are :

16. (i) Now, we will run the following command to get the energies:
grep -i Energy model-default.log
(ii) The results that I got are:
(v) As the query.B99990003.pdb has the lowest energy of 1393.8510, therefore, it is the best model.
17. (i) Generating the cartoon of the Third Model (query.B99990003.pdb) with the help of the following: Show -> As -> Cartoon

(ii) Generating the molecular surface of the Third Model (query.B99990003.pdb) with the help of the following: Show -> As -> Surface

(iii) Generating the Electrostatic surface of the Third Model (query.B99990003.pdb) with the help of the following: Actions→ Generate→
Vacuum Electrostatics→Electrostatic Potential

18. (i) The ‘2mbs_cleaned.pdb’ and ‘query.B99990003.pdb’ were opened in PyMol.
(ii) The Superposition was done with the help of: Action → Align→ to Molecule → 2mbs_cleaned.pdb

(iii) The RMSD came out to be 0.177
SECTION-4: DOCKING
Q19. (i) Opened the website (PrankWeb: https://prankweb.cz/) and uploaded the ‘model_cleaned.pdb’ file to identify potential binding pockets
for binding.

Q20. (i) Firstly, I removed the Heteroatoms with the help of ‘cleanpdb.sh’ to get ‘model_final.pdb’.
(ii) Then, I opened the molecule with the help of Autodocktools by: File→ Read Molecule→ model_final.pdb
(ii) Then, I opened the ligand in Autodocktools with the help of: File→ Read Molecule→ ligand1.pdb

(iii) Then, I did: Ligand→ Input→ Choose→ Ligand→ Click on Select Molecule for Autodock4→ Okay
(iv)Then, I saved it with the help of Ligand→ Output→ Save as “.pdbqt”
(v) Then, I did: Grid→ Macromolecule→ Choose→ Protein→Select Molecule→Save it as “model_final.pdbqt”
(vi) Then, I did: Grid→Set Map Types →Choose Ligand → Select Ligand
(vii) Then, I did: Grid → Grid Box → Spacing (1A) → Setting the size to engulf the whole protein → File → Close saving current.
(viii) Then, I did: Grid → Ouput → Save as ‘gpf’ → Saved as protein.gpf file extension
(ix) Then, I ran Autogrid by: Run → Run Autogrid → Gave log file : lig1.glg → In Bottom pane, gave lig1.glg between -l and s → Launch
(Ran successfully)
(x) The, I did: Docking → Search Parameter → Genetic Algorithm → Default → Accept
(xi) Then, I did: Docking →Macromolecule → Rigid filename → Select protein.pdbqt
(xii) The, I did: Docking → Ligand → Choose → Select ligand1.pdbqt → Accept
(xiii) Then, I did: Docking → Output → Lamarckian GA → lig1.dpf → Save
(xiv) Then, I did: Run → Autdock → Log File: lig1.dlg → In Bottom Pane, gave lig1.dlg between -l and s → Launch (Ran successfully)
(xv) Then, I did: Analyze → Docking → Open → lig1.dlg
(xvi) Then, I did on Terminal:
grep -i Binding lig1.dlg
(xvii) The results that I got are:

Q21. (i) Took the coordinates of the best conformation in a new file named ‘best_conformer.pdb’ and opened it in Gedit.
(ii) Made the following changes in it:
Changed “DOCKED: ATOM” TO “HETATM” with the help of ‘:g/ATOM /s//HETATM/g’ in ViEditor
Removed all the lines not having “ATOM” in it
(iii) Now, made another file named “complex.pdb” and first copied “model_final.pdb” coordinates into it and then “best_conformer.pdb”
coordinates into it.
(iv) Saved it and opened it in Chimera to observe the interaction which is as follows:
Estimated Free Energy of Binding for Ligand 1

(v) To see the 2D Ligand-Receptor Interaction Diagram, I did: Discovery Studio → Receptor-Ligand Interactions → View Interactions →
Show 2D Diagram. The result that I got is:
Receptor-ligand interaction as seen in Chimera
2D Receptor-Ligand Interaction Diagram as seen in Discovery Studio

Q23. (i) I first generated the “ligand1.pdbqt” file with the help of the following command:
obabel -ipdb ligand1.pdb -opdbqt -O ligand1.pdbqt --partial charges gasteiger
(ii) Then, I generated a config file with the following parameters:
(iii) Then, I ran Vina with the help of the following commands:
vina --config config.txt --ligand ligand1.pdbqt --out lig1.pdbqt
(iv)The results that I got are:

(v) Now, we will visualize the result in Autodock by: File→ Read Molecule → model_final.pdbqt→ MS.
(vi) Now, click on Analyze→ Docking→Open Autodock Vina→ Select lig1.pdbqt→Multiple Molecules → Okay.
(vii) The result that I got is:

DOCKING#MOLECULARSIMULATION#ENERGYMINIMISATION

Recommended

Recommended

More Related Content

Similar to DOCKING#MOLECULARSIMULATION#ENERGYMINIMISATION

Similar to DOCKING#MOLECULARSIMULATION#ENERGYMINIMISATION (20)

Recently uploaded

Recently uploaded (20)

DOCKING#MOLECULARSIMULATION#ENERGYMINIMISATION