Vol 4, Issue 3, 2016 ISSN - 2321-4406

Tutorial Article

A HANDBOOK ON PROTEINǧLIGAND DOCKING TOOL: AUTODOCK4

LOKESH RAVI, KANNABIRAN K*

Department of Bio-Medical Sciences, School of Biosciences and Technology, VIT University, Vellore - 632 014, Tamil Nadu, India.
Email: [email protected]
Received: 03 May 2016, Revised and Accepted: 21 May 2016

Keywords: AutoDock 4, Tutorial article, Protein-Ligand Docking, autodock.scripps.edu/). Run “mgltools_win32_1.5.6_Setup.exe” file
AutoDock Installation and leave al the settings default and the installation folder should not
be altered. Finish the installation with the default settings. Once the
INTRODUCTION
installation is finished, “AutoDockTools-1.5.6.exe” will be executed by
AutoDock is an automated suite of protein-ligand docking tools. default. Close the application and start the “AutoDockTools-1.5.6.exe”
AutoDock tools are abbreviated as ADT. It is designed to predict the once again. Once the application is opened for the second time, a new
protein interactions with small molecules such as drug molecule and folder “.mgltools” will be created automatically in the users folder
substrate. The application of this tool is immense, ranging from structure- (C:\Users\Guest\.mgltools).
based drug design, lead molecule optimisation, protein-ligand docking,
protein-protein docking, analysis and validation of mechanism of action To successfully run the AutoDock software, the protein and ligand
of drug molecules, etc., AutoDock has two versions, namely, AutoDock4 structures in their “.pdb” format have to be present within the “.mgltools”
and AutoDock Vina. In this tutorial, the focus is on AutoDock4. folder. The “.mgltools” folder has a subfolder “1.5.6.” Create a new folder
within “1.5.6” folder, to use as a working folder (e.g.: “WorkFolder”) as
AutoDock4 analyzes the interactions of ligand molecules at the given in Fig. 1A. The “.pdb” files of the protein and ligand to be studied
specified target site of the protein. The users can define this specific must be pasted within this working fold er. For easier use of the tool, the
target sites with the use of GridBox. AutoDock4 has two key programs default startup directory for AutoDock has to be changed to a working
to be executed, i.e., Autogrid4 and AutoDock4. Autogrid4 prepares a folder.
grid map of the amino acids presents within the GridBox defined by
the user. AutoDock4 then analyzes the interactions of those amino acids To setup the default folder:
with the ligand molecule.
File » Preferences » Set
AutoDock4 has options to set the protein molecule to be rigid or flexible.
While, the ligand molecule is automatically annotated for its rotatable and ª Startup Directory » “C:\Users\Guest\.mgltools\1.5.6\
non-rotatable bonds and determine the flexibility. In this tutorial, the docking WorkFolder”
procedure is displayed for rigid macromolecule. Rigid macromolecule
docking is faster than that of the flexible macromolecule docking. ª Make Default » Dismiss

AutoDock4 predicts the free binding energy with a scoring function Next, run the “autodocksuite-4.2.5.1-i86Windows.exe” file and select
based on the AMBER force field and linear regression analysis, a custom folder for this. At the end of the installation, there will be two
additionally by referring to a large set of library data of known protein- execution files created in the selected custom folder (i.e., AutoDock4.
ligand interactions with their inhibition constants that were used in exe & autogrid4.exe). These two files have to be moved into “1.5.6”
AutoDock3. It is found that the standard error in free binding energy in folder within the “.mgltools” folder [C:\Users\Guest\.mgltools\1.5.6]
AutoDock4 is approximately 2.5 kcal/mol. highlighted in Fig. 1B. At this stage, the software is ready to be
executed.
In this tutorial, a basic protocol to use AutoDock4 to analyze interactions
of selected protein and ligand molecule is discussed. This protocol PREPARING THE PROTEIN MOLECULE
is presented to aid students and research scholars those who are Protein molecules can be downloaded from Protein Data Bank website
interested in using AutoDock4 for learning and or research purpose. (www.rcsb.org). The downloaded protein structure in their “.pdb”
This protocol is considered to be adequate and effective by the authors, format has to be edited to remove the non-aminoacid residues, such
to use AutoDock4 for protein-ligand docking analysis. as water molecules, ions, ligands that are in the complex. These can be
removed using either PyMol software or WordPad. This has to be done,
The discussed protocol can be used to study the interactions of selected
since, these molecules will interfere with the interaction between the
ligand molecule (drug molecule) with chosen protein targets. Users,
target molecule and protein in AutoDock.
however, have to do a little preparative work on the protein molecule pdb
file. Downloaded 3D structure of proteins from RCSB website has to be
Once the “.pdb” file is downloaded, right-click the file and open it with
edited before docking in AutoDock4. Although a small discussion on the
WordPad. Scroll down to the end of the document and delete all the
preparation of protein molecules is presented in this tutorial, users are
lines that begin with “HETATM” and “CONNECT,” these are the atoms
advised to do further reading in the preparation of protein structures.
that do not belong to amino acids and their interaction with the amino
acids, respectively. The last line of the document should contain a like
INSTALLING AUTODOCK
begins with “TER” (e.g.: “TER 8910 SER B 557”) this indicates the end of
Download the installation files “autodocksuite-4.2.5.1-i86Windows.exe the protein chain. Once this is done, save the document, now it is ready
and mgltools_win32_1.5.6_Setup.exe” from AutoDock website (http:// for AutoDock analysis.
 Ravi and Kannabiran
Innovare Journal of Medical Science, Vol 4, Issue 3, 1-6

Fig. 1: Contents of the folder C:\Ussers\Guest\.mgltools\1.5.6\

EXECUTING AUTODOCK To initialize molecules

AutoDock software calculates and predicts the interaction between File » Read Molecule
the ligand molecule and protein molecule based on predefined
parameters. To be precise, the interactions between the molecules ª Select Protein File (“.pdb” file)
will be calculated at a user specified region in the protein. This region
can be defined by users, using the Grip map option. Ultimately, the Set the protein view to Ribbon and Surface view (Fig. 3A)
software predicts the interaction and binding energy of the ligand
molecule and the amino acids present within the GridBox only. Thus Edit » Hydrogens » Add
setting, the GridBox at the binding site or active site or other essential
ª Polar Only » OK
regions of the protein is important. Before executing the AutoDock,
the “.pdb” files of the protein and ligand have to be moved into the
Edit » Hydrogens
“WorkFolder.” Fig. 2 gives a graphical representation of contents of
“WorkFolder” before and after executing AutoDock. The “WorkFolder”
ª Merge Non Polar » Continue [Click ‘CONTINUE’ for the
should contain only the “.pdb” files of protein and ligand at the
    Warning]
beginning.
Edit » Charges » Add Kolman Charges
Analysis in AutoDock can be divided into following categories,
(1) Initializing molecules; (2) Running AutoGrid; (3) Running AutoDock;
ª OK
(4) Analyzing Interaction energy.
Initializing molecules File » Save » Write PDB
Initializing the molecule mainly includes addition of hydrogen atoms
and addition of Kolman charge to the protein molecule. While for the ª Sort Nodes (Check) » OK » (Overwrite) YES
ligand molecule, addition of Gasteiger charge, identifying aromatic
carbons, detecting rotatable bonds, and setting TORSDOF value. The Ligand » Input » Open
protein has to be initialized manually, while the ligand is automatically
initialized when opened in the tool. ª (Change view to all files) Select Ligand File (“.pdb” file) » OK

Once the protein molecule is opened, it is important to change the view Ligand » Output » Save as PDBQT
of the protein. It will be in “line view” by default. Enable the “Ribbon
view” and “Surface view” as given in Fig. 3A. It is essential that the ª OK
protein is in “Ribbon view,” failing to do so, might result in errors in the
later steps. The surface view makes it user-friendly to set the GridBox Executing AutoGrid4
in the later steps. Fig. 3 highlights all the important menu options that AutoGrid has to be executed, to define the region/area in the protein
will be used in the given protocol. to be analyzed for the interaction with the ligand molecule. In general,

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Innovare Journal of Medical Science, Vol 4, Issue 3, 1-6

Fig. 2: Contents of WorkFolder before and after executing AutoDock (A: Before executing AutoDock, B: After executing AutoDock)

Fig. 3: Key options to be used in the AutoDock window

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Innovare Journal of Medical Science, Vol 4, Issue 3, 1-6

the region of interaction could be identified using prediction tools such ª File name “grid.gpf” » SAVE
as Q-Site finder and MetaPocket to identify the binding pockets on the
surface of the proteins. Then, the GridBox is set in AutoDock to cover Run » Run AutoGrid (Fig. 5A)
the identified binding sites.
ª Select Program Pathname: ‘autogrid4.exe’
AutoDock only analyzes the interactions of ligand molecule and the
amino acids that are present within the GridBox. So setting up, the ª Select Parameter Filename: ‘grid.gpf’
GridBox is a crucial step.
ª AutoFill for Log Filename: ‘grid.glg’
The grid options menu has two fields as shown in Fig. 4. The size of the
ª LAUNCH
GridBox can be increased or decreased using the number of points in
X/Y/Z dimension scroll option (Fig. 4A). The position of the GridBox
Executing AutoDock4
can be adjusted to cover the binding site or binding residues, using the Once AutoGrid is successfully completed, AutoDock can be executed.
Center GridBox field, that moves the GridBox in the X/Y/Z axis (Fig. AutoDock calculates the interactions of the amino acid within the
4B). GridBox and ligand molecule.

To Run AutoGrid To Run AutoDock

Grid » Macromole » Choose Docking » Macromolecule » Set Rigid filename
ª Click (Protein) » Select Molecule » OK » SAVE
ª Select ‘Protein.pdbqt’ » Open
Grid » Set Map types » Choose Ligand Docking » Ligand » Choose

ª Click (Ligand) » Select Ligand ª Click ‘Ligand’ » Select Ligand

Grid » GridBox ª Accept

ª SET the BOX Docking » Search Parameters » Genetic Algorithm

ª File » Close Saving Current ª Accept

Grid » Output » Save GPF Docking » Output » Lamarckian GA

Fig. 4: Grid options in AutoDock. (A: To adjust the size of the GridBox, B: To adjust the position of the GridBox)

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Innovare Journal of Medical Science, Vol 4, Issue 3, 1-6

Fig. 5: Panels to execute autogrid4.exe and autodock4.exe (a: Panel to execute autogrid4.exe, b: Panel to execute autodock4.exe)

ª Save file as ‘dock.dpf’ Analyze » Macromolecule » Choose

Run » Run AutoDock (Fig.5B) ª Click ‘Protein’ » Select Macromolecule

ª Select Program Pathname: ‘autodock4.exe’ Analyze » Conformations » Play, Ranked By Energy (Fig. 6A)

ª Select Parameter Filename: ‘dock.dpf’ ª Click on the ‘&’ Button (Open panel to change play options)

ª AutoFill for Log Filename: ‘dock.dlg’ Set Play Options (Fig.6B)

ª LAUNCH ª Check ‘Build H-Bonds’

Analyzing interaction energy ª View the hydrogen bonds formed (Fig.6C)

Once AutoDock4.exe is successfully executed. The result will be given
as the ten best confirmations. These can be viewed in the analyze ª Check ‘Show Info’
options. The confirmations can be viewed in the order of their
ª View the Interaction Energy (Fig. 6D)
free energy binding, by choosing the “Play, ranked by energy” option.
ª Build Current Write Complex
Analyzing the result takes a few steps that is given as a graphical
representation in Fig. 6. The panels a, b, c and d will open one after the
ª Save as ‘Result.pdb’ ” Save
other, in the same order as given. The ten conformations can be viewed
by changing the conformations number in the panel A (Fig. 6A). The CONCLUSION
interaction energy of the given conformation can be viewed in the panel
D (Fig. 6D). The number of hydrogen bonds formed between the ligand The presented protocol to use AutoDock4 is one of the many ways to
and protein can be viewed in the panel C (Fig. 6C). use ADT. The authors find this to be a simple and beginners protocol
to learn AutoDock4. This protocol is designed based on the authors
To Analyze Interaction energy experience in handling of the ADT and no troubleshooting is required, if
the protocol is followed as such. If error messages do appear, then kindly
Analyze » Docking » Open start over the protocol, by editing or changing the protein molecule, as it
is most likely that, the error could originate from the protein molecule
ª Select ‘dock.dlg’ » Open structure. Thus, it is advised to the users, to do further reading in editing
and/or preparation of the protein molecule. This protocol is best suited
ª Assign Ligand New Name » OK for teaching and learning of AutoDock4. Furthermore, it is a beginner’s

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Fig. 6: Panel to view the interaction energy of the top ten conformations

protocol for researchers to analyze simple interactions of ligand molecule from the AutoDock output. The users are recommended to use PyMol
and target protein. Since AutoDock4 is only suitable for selective site software to view the final result.pdb file since it is easier to visualize.
docking, if the users do not know the region of interest or binding site, the
results might be irrelevant. Hence, the users are advised to identify the ACKNOWLEDGMENT
region of interest in the given protein using binding pocket identifiers,
active site prediction tools, etc. Researchers have to identify the region This protocol is presented based on the author’s experience and is
of binding or docking, with help of several online tools that predicts the not an official standard. The authors do not claim any rights over the
binding site, active site, binding pockets, etc. Once the given protocol is tool or its functions. This protocol is designed purely for educational
completed, the energy values and inhibition constant can be obtained intentions and to help students learn AutoDock4.