Illustrated Step-By-Step Protocol To Perform Molecular Docking

Qarshi Brands (Pvt.) Ltd.
HANDS-ON WORKSHOP
15. 04. 2023
Illustrated step-by-step protocol

to perform molecular docking
Dr. Shafi Ullah Khan

Manager
Product and Process Innovation Department
Qarshi Brands Pvt. Ltd.
Illustrated Step by step protocol to perform molecular docking: human

estrogen receptor complex with 4-hydroxytamoxifen as a case study
Background and Introduction:
Molecular docking is one of the most frequently used methods in structure-based drug
design, due to its ability to predict the binding-conformation of small molecule ligands
to the appropriate target binding site (1). Molecular docking helps in moving the process
of computer- aided drug designing faster and provides every conformation possible
based on the protein and ligand molecule (2). Docking has a huge advantage when it
comes to the study of protein interactions. To date many molecular docking protocols
has been reported but to best knowledge no illustrated protocol is reported which
mentioned from basic start to end analysis of result for any specific case (3). In this
protocol we used very simplest possible approach for performing molecule docking
study. We designed this protocol for very novice user who does not have any prior
experience of molecular docking study. To make this tutorial applicable and generalize,
we started from downloading and installation of all freely available pre-requisite
software which was followed by subsequent illustrated step by step methodology to
make possible execution of each step.
Basic Idea for selecting the Human estrogen receptor Alpha ligand-binding domain in
Complex with 4-Hydroxytamoxife is due to fact that this receptor plays important role
in physiological development and function of a variety of organ systems to varying
degrees, including the reproductive, central nervous, skeletal, and cardiovascular
systems (4). We believe that this illustrated step by step tutorial will be helpful for all
novice user who are interested in drug designing, Insilco screening, virtual screening
and binding of ligand with protein structure. Advice to reader and user is that, follow
each and every-step to execute this tutorial your-own and then change accordingly for
your protein or target of interest (5,6).
If for some reason, said protocol is not applicable for your target of interest, try to figure
out the difference in protein by doing toughly literature review of your target and note
down basic architecture and assembly of your target, amino acid residues involved the in
active site of target. Sometimes active site also comprised of metals, cofactor or
conserved water molecule which need to be treated very carefully (6).
Before setting to implement this tutorial for any target or protein or receptor of interest,
keep in mind that this tutorial is very basic, to make new user used to different task
execution in Molecular docking by using Autodock Tool (7), autogrid4, sutogrid4 (8)and
discovery studio (9). We strongly believe that this tutorial will make anyone able to do
molecule docking by themselves even if he/she does not have any prior experience.
REQUIREMENTS
1. Window 7, 8 or 10
2. Freely available software’s
3. MGL tools
4. Binary files of Autodock and Autogrid
5. Discovery Studio Visualizer
MGL tools
Downloading MGL tools

Go to http://autodock.scripps.edu/downloads
or
https://ccsb.scripps.edu/mgltools/downloads/
Click on the mgltools_win32_1.5.6_Setup.exe as shown

Installation of MGL tools (autodock Tool)
After Downloading the mgltools_win32_1.5.6_Setup file double

click in an order to start installation.
Click Next
Wait till pop-up displays on screen. Click OK after completion.

Click Finish.
Binary files of Autodock andAutogrid
Downloading Binary files of Autodock andAutogrid

Go to http://autodock.scripps.edu/downloads
Or
https://autodock.scripps.edu/download-autodock4/
Click on Proceed to the Download page
Scroll Down in open tab and click on Windows/Mac OS X to download as

shown below
Installation of Binary file (AutoDock andAutoGrid)

Double click on downloaded file named autodocksuite-
4.2.6.i86Windows
*Note the installation Directory
Click Close after installation complete

Discovery Studio Visualizer

Downloading Discovery Studio Visualizer
Go to
https://discover.3ds.com/discovery-studio-visualizer-
download#_ga=2.211774352.420850172.1648320530-9a108df0-2bf9-11ec-
918e-af3b0b5a0655
fill in the form as per template and submit .
After successful submission you will

need to download DS from link:
Download the version 32 bit or 64 bit
depend on OS window in your system.
In our case, it is
DS Visualizer Client (Windows 64
bit)
https://discover.3ds.com/discovery-
studio-visualizer-download-thank-you
Installation of Discovery Studio Visualizer

Double click on downloaded file i.e DS2017R2Client.exe is this
tutorial
Specify the Directory

C:\Program Files\BIOVIA\Discovery Studio 2017R2\
Click
Next
Then Click on Install
Click Finish
Now Three basic tool (AutoDockTools-1.5.6, AutoDockSuit and Discovery

Studio) are ready for further usage
After Setting up the above-mentioned software

successfully
Next Step is
Retrieving Ligand and Protein files from major databases
Retrieving target protein from major protein databases
i.e RCSB Protein Data Bank
Go to
https://www.rcsb.org/
Type the query protein or enzyme in search

In this case we used 3ERT as pdb code of (Human
Estrogen Receptor Alpha Ligand- Binding Domain In
Complex With 4-Hydroxytamoxifen)
Click on 3ERT
Click first on Download Files on right hand side and then in

download menu click on PDB Format.
It will download 3ert.pdb file in download directory
Open 3ert.pdb (can be any other protein in different

case) in Discovery Studio Visualiser
Go to View > Hierarchy or simply Press Control+H

It will open Hierarchy window on left side of graphical
window as shown below and highlighted in Red rectangle
You will see that there are three A, correspond to different entities like
First one is comprised of amino acid (Leu306-Pro552), second one is Co-
crystal ligand (4-OHT600) and third one is comprised of H2O (HOH1-
HOH79). Using the inbound ligand one can easily find out the active site
dimension and coordination for Grid box generation.
Getting XYZ dimension and coordination of Co-crystal ligand (4-

OHT600)
Expand 2nd A in hierarchy window and right click on 4OHT600 will
pop-up small window as shown below. At the end of the pop-up
window Click on Attributes of OHT600
Norte down the XYZ coordinates from this small pop-up

window. In this case of OHT600 it is (X=31.574552,
Y=1.590379 and Z=25.599483)
After noting down of XYZ dimension and coordinate, we need to

remove water and extract co-crystal ligand (in this case OHT600) for
redocking and optimization of docking protocol.
Select 2nd A chain in hierarchy window and make cut paste in new
window of Discovery studio.
Click on File on left side and Save as lligand.pdb file in working
directory (One can save with different name to differentiate different
ligands from one another)
Note: Ligands can be sketch using different software like

MarvinSketch, ChemDraw as well various online servers frequently
using one is PUBCHEM online server database
Delete the 3rd A chain in hierarchy window to delete all water

molecule (Some time in protein, few conserve water molecules
need to retain which are involved in stabilization of ligand-protein
complex).
Now this protein is free from heteroatoms i.e water and co-crystal
ligands.
Click on File on left side and save prepared structure as

protein.pdb file in working directory. (One can also save with
different name to differentiate different protein from one another)
Check and confirm that the protein.pdb and ligand.pdb files are
present in the specified folder (in this case Tutorial)
After successfully completion of downloading ligand and protein file

Next step is
Preparation of pdbqt format file of ligand and protein usingAutodock
tool
Preparing PDBQT format for Protein and ligand (Protein.pdbqt,
Ligand.pdbqt), Grid and Docking Parameter file (a.gpf and a.dpf) using
AutoDock 4.2
• Open AutoDockTools-1.5.6 (previously installed)

AutodockTools shortcut should present on desktop
AutoDock Tools Display

Preparation of Protein
Preparation of Protein.pdbqt file
Open File
Read Molecule
Go to specifies folder where
preotin.pdb was saved Select and
Open protein.pdb
Protein in AutoDock tool display
Click on Edit
Click on Hydrogens
Click on Add
Click Polar Only in next

pop-up window
Click OK
• Again go to Edit
• Click Charges
• Add Kollman Charges
• Click OK
• Open Grid
• Click on Macromolecules
• Click on Choose
Click protein in pop-up window

Click Select Molecule
Click OK if waring window pop-up

Save protein.pdbqt in specified folder
After saving macromolecule in protein.pdbq format (it

gets coloure change from pink to grey) as shown below
After saving protein.pdbqt file in specified folder . next step is the

preparation ligand.pdbqt file
Note: (Be careful with case sensitive letter and naming of protein naming
as it may affects the follow-up commands execution).
Close and open AutodockTool again to prevent any misunderstanding
Preparation of Ligand
Preparation of Ligand.pdbqt file
Open Ligand
Click Input
Click Open
Change file format from .pdbqt to .pdb in

next pop-up window
Select ligand
Click OPEN
Click OK in pop-up window of summary of ligand

Again Open Ligand

Click Torsion Tree
Click Detect Root
Again click Ligand

Click Torsion Tree
Click Set Number of Torsions
Set number of active torsions between 1 to 8

(in this we select 8 i.e maximum no. of torsion)
Click Dismiss
Again Open Ligand

Click Aromatic Carbons
Click Aromaticity criterion
Click OK (* If ‘Enter angle in Degrees: 7.5’)

Again Open Ligand

Click Output
Click Save as PDBQT
Save Ligand file as ligand.pdbqt in specified folder

* In the same folder and working directory
where protein.pdbqt was saved.
After preparation of protein.pdbqt and ligand.pdbqt file next step is the Preparation of Grid
Parameter File (protein.gpf)
Open protein.pdbqt and ligand.pdbqt in AutodockTool as shown in figure

Open Grid
Click Set Map Types
Click Choose Ligand
Click Ligand
Click Select Ligand
Again Open Grid

Click Grid Box
Use Change X,Y,Z dimension as 40x40x40 with spacing 0.375
Center Grid Box xyz 31.570, 1.590, 25.590
Note these XYZ dimension and coordination, we gotten previously
described from attributes of co-crystal ligand.
Note: This center Grid Box can be changed according to the active
site of different protein.
After selection of predefined XYZ dimensiona dn coordination.

Go to File and select “Close saving current”. It will the savethe
adjusted dimension.
Again Open Grid

Click Output
Click Save GPF
Name the File name as protein.gpf in specified folder where

protein and ligand .pdbqt files were saved
Preparation of Docking Parameter File (ligand.dpf)

Open Docking
Click Macromolecules
Click Set Rigid Filename
Go to working directory or folder

Select protein.pdbqt
Click Open
Again go to Docking
Click Ligand
Click Choose
Click Ligand
Click Select Ligand
Again go to Docking
Click Search Parameters
Click GeneticAlgorithm
Click Accept
Click Accept (*Using Default but we can change no. of GA runs)

Again Docking
Click Docking parameters
Go to Docking>Docking Parameter
and Click Accept (*Using Default)

in pop-up window
Again go to docking
Click Output
Click LamarkianGA(4.2)
Name the File name as ligand or else you wish in this case
we save as ligand.dpf file
Note: Save protein.gpf and ligand.dpf in the same folder
or working directory where protein.pdbqt and ligand
.pdbqt and protein.gpf files were already saved.
Now along with protein.pdb and ligand.pdb, four

additional files protein.pdbqt, ligand.pdbqt,
protein.gpf and ligand.dpf will be present in the
working directory directory or folder as shown below
Setting AutoDock for running (Simplified approachfor

novice users)
Locate the insatlledAutodock and Autogrid in default
installation directory.
Default installation directory could be “C:\Program Files
(x86)\The Scripps Research Institute”
Copy that Autodock4.exe and autogrid4.exe files from
Installation folder into the working directory (In this case is
working directory is Desktop\Tutorial”
After the Preparation of Required files such as

.pdbqt files of both protein and ligands
.gpf file of protein
.dpf file of ligand
And copying binary file of Autodock4.exe and autogrid4.exeinto
the working directory Next step is
running Autogrid4 and autodock4
Using Command Prompt (command line in window)for

Molecular Docking
Open CMD, From Window start search for CMD and click to open
Open CMD
Go to working directory where required files are presentusing

commod i.e “cd workingdirectoy address” in this case
cd C:\Users\Shafi_Khan\Desktop\Tutorial
Now we are in the workshop directory where other pre-requisite files are
presents
Running of Autogrid for .gpf to .glg conversion (use below commond)
autogrid4.exe -p protein.gpf -l protein.glg
Note: Depend on protein name and working directory this need to change
accordingly”
*Blinking at the start of line indicated that process is running
Normally, it takes few minutes to complete depend on the performance of
system
When processing is finished the curser will shift to the

specified working directory location
Running Autodock for .dpf to .dlg conversion (use below

commond)
autodock4.exe -p ligand.dpf -l ligand.dlg
*Blinking at the start of line indicated that process is running
It will take quite some time (from minutes to hours) to complete
depend on the performance of system
Command is in progressing state
When processing is finished, the cursor will go forward to

the working directory location.
Command is in working directory

After completion of autodock and autogrid processing, both

protein.glg as well as ligand.dlg along with map files for each
atoms could be found in working directory folder as shown below
Results Analysis
Analysing results and Retrieving interaction in

Ligand-protein complex .pdb
Open AutoDock
Click Analyze
Click Docking
Click Open
Select ligand.dlg
Click Open
Click OK
Open Macromoleule
Click Analyze
Click Macromoleule
Click Open
Select protein.pdbqt
Click Open
Again go to Analyze
Click Conformations
Click Play
Click in sequential steps from1 to 3

1. Click on “&” sign, followed
2. Click show info and then
3. Click on Write current
Above sequential will generate the pdbqt file of corresponding
pose (in this case pose 1 is the best having lowest binding free
energy of -11.33) in default startup directory of Autodock tool
as shown in below.
Save as ligand_1.pdbqt in working directory (can be named
differently depend on the conformation with best binding
energy)
Note: Lower the binding energy better would be the ligand-

protein complex
Click “ ” play sign to observe each conformation or pose from 1 to

10 and note down the difference in free binding energy for each
conformation.
Click on Write Current to generate separate pose having configure
coordinates.
Analysis of Nonbonding Interactions

Open protein.pdbqt in Discovery Studio
Drag and drop ligand_1.pdbt from working directory into

DS graphical window as shown in figure
Click Scripts in Discovery studio

Click Ligand Interactions
Click Show Ligand Binding SiteAtoms
Right click on graphical window and

select Labels and Click Add
Select Object:AminoAcid
Select Attributes: 3 Letter & ID#
Click OK
Saving as Image files

Go to File and then SaveAs
Change file format form Discovery

Studio files to Image Files
Name as ligand_1_3D
Click Save and then OK to Save in working directory
Click OK
Final Image in 3D formate
On left hand side in the ligand interaction window click on

show 2D interaction will give the 2D image of binding
interaction between the ligand and protein
Analyse critically the binding interactions within 4OHT re-

docked one and co-crystal one and check whether there is any
difference within these two or not.
If there is no significant difference between these two,
decipher that the followed docking protocol seems deem
enough for further molecular docking of other compounds.
References
1. Yuriev E, Agostino M, Ramsland PA. Challenges and advances in computational
docking: 2009 in review. Journal of Molecular Recognition.2011 Mar;24(2):149-64

2. Yuriev E, Holien J, Ramsland PA. Improvements, trends, and new ideas in molecular docking:
2012–2013 in review. Journal of Molecular Recognition. 2015 Oct;28(10):581-604.
3. Rizvi SM, Shakil S, Haneef M. A simple click by click protocol to perform docking: AutoDock
4.2 made easy for non-bioinformaticians. EXCLI journal. 2013;12:831.

4. Coward P, Lee D, Hull MV, Lehmann JM. 4-Hydroxytamoxifen binds to and deactivates the
estrogen-related receptor γ. Proceedings of the National Academy of Sciences. 2001 Jul
17;98(15):8880-4.
5. Erickson JA, Jalaie M, Robertson DH, Lewis RA, Vieth M. Lessons in molecular recognition:
the effects of ligand and protein flexibility on molecular docking accuracy. Journal of medicinal
chemistry. 2004 Jan 1;47(1):45-55.
6. Ferreira L, dos Santos R, Oliva G, Andricopulo A. Molecular docking and structure- based drug
design strategies. Molecules. 2015;20(7):13384-421.
7. Morris GM, Huey R, Olson AJ. Using autodock for ligand‐receptor docking. Current protocols
in bioinformatics. 2008 Dec;24(1):8-14.
8. Goodsell DS, Morris GM, Olson AJ. Automated docking of flexible ligands: applications of
AutoDock. Journal of Molecular Recognition. 1996 Jan;9(1):1-5.
9. Biovia DS. Discovery studio visualizer. San Diego, CA, USA.2018.


Illustrated Step-By-Step Protocol To Perform Molecular Docking

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Qarshi Brands (Pvt.) Ltd.

Illustrated step-by-step protocol

Dr. Shafi Ullah Khan

Illustrated Step by step protocol to perform molecular docking: human

Downloading MGL tools

Click on the mgltools_win32_1.5.6_Setup.exe as shown

Installation of MGL tools (autodock Tool)

After Downloading the mgltools_win32_1.5.6_Setup file double

Wait till pop-up displays on screen. Click OK after completion.

Downloading Binary files of Autodock andAutogrid

Scroll Down in open tab and click on Windows/Mac OS X to download as

Installation of Binary file (AutoDock andAutoGrid)

Click Close after installation complete

Discovery Studio Visualizer

After successful submission you will

Installation of Discovery Studio Visualizer

Specify the Directory

Then Click on Install

Now Three basic tool (AutoDockTools-1.5.6, AutoDockSuit and Discovery

After Setting up the above-mentioned software

Type the query protein or enzyme in search

Click first on Download Files on right hand side and then in

Open 3ert.pdb (can be any other protein in different

Go to View > Hierarchy or simply Press Control+H

Getting XYZ dimension and coordination of Co-crystal ligand (4-

Norte down the XYZ coordinates from this small pop-up

After noting down of XYZ dimension and coordinate, we need to

Note: Ligands can be sketch using different software like

Delete the 3rd A chain in hierarchy window to delete all water

Click on File on left side and save prepared structure as

After successfully completion of downloading ligand and protein file

• Open AutoDockTools-1.5.6 (previously installed)

AutoDock Tools Display

Protein in AutoDock tool display

Click Polar Only in next

Click protein in pop-up window

Click OK if waring window pop-up

After saving macromolecule in protein.pdbq format (it

After saving protein.pdbqt file in specified folder . next step is the

Change file format from .pdbqt to .pdb in

Click OK in pop-up window of summary of ligand

Again Open Ligand

Again click Ligand

Set number of active torsions between 1 to 8

Again Open Ligand

Click OK (* If ‘Enter angle in Degrees: 7.5’)

Again Open Ligand

Save Ligand file as ligand.pdbqt in specified folder

Open protein.pdbqt and ligand.pdbqt in AutodockTool as shown in figure

Again Open Grid

After selection of predefined XYZ dimensiona dn coordination.

Again Open Grid

Name the File name as protein.gpf in specified folder where

Preparation of Docking Parameter File (ligand.dpf)

Go to working directory or folder

Click Accept (*Using Default but we can change no. of GA runs)

and Click Accept (*Using Default)

Now along with protein.pdb and ligand.pdb, four

Setting AutoDock for running (Simplified approachfor

After the Preparation of Required files such as

Using Command Prompt (command line in window)for

Go to working directory where required files are presentusing

When processing is finished the curser will shift to the