protocol

Computational proteinligand docking and virtual

drug screening with the AutoDock suite
Stefano Forli, Ruth Huey, Michael E Pique, Michel F Sanner, David S Goodsell & Arthur J Olson

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA. Correspondence should be addressed
to A.J.O. ([email protected]).

Published online 14 April 2016; doi:10.1038/nprot.2016.051

Computational docking can be used to predict bound conformations and free energies of binding for small-molecule ligands
to macromolecular targets. Docking is widely used for the study of biomolecular interactions and mechanisms, and it is applied
to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of
thousands of compounds. This protocol covers the docking and virtual screening methods provided by the AutoDock suite of
programs, including a basic docking of a drug molecule with an anticancer target, a virtual screen of this target with a small
ligand library, docking with selective receptor flexibility, active site prediction and docking with explicit hydration. The entire
2016 Macmillan Publihers Limited. All rights reserved.

protocol will require ~5 h.

INTRODUCTION
Computational docking is widely used for the study of protein based on empirical free energies of binding, is used to score poses
ligand interactions and for drug discovery and development. quickly at each step of the conformation search.
Typically, the process starts with a target of known structure, Both of these are serious limitations, and users must use
such as a crystallographic structure of an enzyme of medicinal tools such as molecular dynamics or free-energy perturbation
interest. Docking is then used to predict the bound conformation if a more realistic conformational search or energy prediction
and binding free energy of small molecules to the target. Single is necessary. These tools are complementary to computational
docking experiments are useful for exploring the function of docking methods, as docking methods generally search a larger
the target, and virtual screening, in which a large library of conformational space, but more advanced methods can predict
compounds are docked and ranked, may be used to identify conformation and energy more accurately within a local area of
new inhibitors for drug development. the conformational landscape.
AutoDock is a suite of free open-source software for the Advanced docking methods may be used to improve results
computational docking and virtual screening of small molecules in cases in which the limitations of requiring a rapid method
to macromolecular receptors. The suite currently includes several for energy evaluation are too restrictive. For instance, many
complementary tools: docking methods use a rigid model for the receptor, which
often leads to improper results for proteins with an appreci-
AutoDock Vina: a turnkey computational docking program able induced fit upon binding. AutoDock includes a method
that is based on a simple scoring function and rapid gradient- for treating a selection of receptor side chains explicitly, to
optimization conformational search1. account for limited conformational changes in the receptor.
AutoDock: a computational docking program based on an In addition, ordered water molecules often mediate interac-
empirical free-energy force field and rapid Lamarckian genetic tions between ligands and receptors, and advanced methods for
algorithm search method2,3. treating selected waters explicitly have been implemented in
Raccoon2: an interactive graphical tool for virtual screening AutoDock. Both of these advanced methods are demonstrated
and analysis4. in this protocol.
AutoDockTools (ADT): an interactive graphical user interface Many reports have compared the performance of popular
(GUI) for coordinate preparation, docking and analysis5. docking methods such as AutoDock (recently reviewed by Sousa
AutoLigand: a program for predicting optimal sites of ligand et al.7). Different methods can achieve different success rates
binding on receptors6. depending on specific targets, but in general they all perform sim-
ilarly when tested on a series of diverse proteinligand complexes:
The AutoDock suite, including source code, is freely available, and they all perform well for the prediction of bound complexes for
it has been widely used in research and drug discovery. drug-sized molecules, with estimates of free energies of binding
with errors of roughly 23 kcal mol1, provided that there is no
Comparisons with other methods significant motion required in the receptor. Better results may be
A variety of academic and commercial methods for computational obtained by tuning the docking method for a particular system
ligand docking are currently available (see Sousa et al.7 for an or moving to more sophisticated and computationally intensive
extensive review of current methods). Most of these methods sim- parameterizations of the system.
plify the problem in two ways to make the computation tractable.
First, the conformational space is reduced by limitations imposed Applications of the protocol
on the system, such as a rigid receptor and fixed bond angles and The AutoDock suite is used in numerous laboratories: a recent
lengths in the ligand. Second, a simplified scoring function, often search on PubMed yielded over 1,000 citations in the past year.

nature protocols | VOL.11 NO.5 | 2016 | 905

 protocol
We have applied the AutoDock suite to a number of different Docking to a collection of different receptor structures that cover
problems in collaboration with researchers, showing some of the expected range of flexibility in the receptor. These may be
the scope of uses. The first versions of AutoDock were used to obtained from multiple structural determinations or simulation.
explore the binding of substrates to the enzyme aconitase8, and Use of explicit receptor side-chain flexibility during docking,
docked conformations were used to help interpret crystallo- if information is available on relevant side chains (described in
graphic density maps and to explore intermediates in the reaction. the protocol).
Results of several similar studies on proteins and their substrates
were reported in the following years911. We have also used the The scoring methods also use a variety of simplifications that
AutoDock suite as a tool for drug design and virtual screening will affect the results. The AutoDock Vina scoring function is
against a number of targets, including HIV protease1214, tuber- highly approximate, with spherically symmetric hydrogen bond
culosis targets15,16 and -secretase17,18. AutoLigand was used potentials, implicit hydrogens and no electrostatic contribution.
to identify druggable sites for stabilizing a proteinprotein It has been demonstrated to perform well with ligands with typi-
interface19, and covalent docking was used to explore covalent cal biological size and composition. The AutoDock force field
inhibitors of protein tyrosine phosphatases20. includes physically based contributions, including a directional
hydrogen-bonding term with explicit polar hydrogens, and elec-
Experimental design trostatics. If these contributions are important in a particular
AutoDock and AutoDock Vina. Two docking methods system, AutoDock would be the appropriate tool. In addition, the
2016 Macmillan Publihers Limited. All rights reserved.

have been developed in parallel, to respond to two different parameterization of the AutoDock scoring function is available
needs. Development began with AutoDock 2,3,5,21,22, and it to the user, to allow tuning for particular systems if desired. For
continues to be the platform for experimentation in docking instance, methods for incorporating explicit solvents and for
methods. AutoDock Vina was developed more recently to fulfill predicting conformations of covalent complexes were developed
the need for a turnkey docking method that does not require by modifying the AutoDock potentials23,24.
extensive expert knowledge from users1. It is highly optimized
to perform docking experiments using well-tested default Raccoon and virtual screening. Virtual screening is rapidly
methods. Both methods are currently freely available. AutoDock becoming the primary application of computational dock-
Vina is fast and effective for most systems, whereas AutoDock ing methods, with many successes in the discovery of new lead
is available for systems that require additional methodological compounds for pharmaceutical development25. The idea is
enhancements. to screen a large library of available ligands to identify a small
Both methods are designed to be generic computational docking subset for purchase and experimental testing.
tools, by accepting coordinate files for receptor and ligand and pre- Raccoon is a graphical user interface that is designed to
dicting optimal docked conformations. Typically, users start with streamline the steps of a virtual screening and analysis of the
receptor coordinates from crystallography or NMR spectroscopy results. These include the following:
and with ligand coordinates generated from SMILES (Simplified
Molecular-Input Line-Entry System) strings or other methods. Automated server connection manager and installation of
Because the search methods are stochastic, a set of optimal docking services (such as AutoDock Vina).
docked conformations is predicted, and then they are typically Ligand library for upload and management of large ligand
clustered spatially to analyze the consistency of the results. collections.
Highly clustered results are an indication that the conforma- Receptor management from multiple targets and flexible
tional search procedure is exhaustive enough to ensure coverage residues.
of the accessible conformational space. Because of the stochastic Graphical interface for docking parameter setup.
nature of the search, the method cannot ensure that a global Graphical management of jobs on computational resources.
minimum has been found. For this reason, it is important to Automated retrieval and preprocessing of results to extract
use re-docking experiments with known complexes of similar features of interest.
conformational complexity to evaluate the docking protocol User-friendly filtering of virtual screening results based on
being used. properties and interactions.
AutoDock and AutoDock Vina currently use several simplifica- Export of filtered results.
tions that affect the results that are obtained. The most significant
simplification is the use of a rigid receptor. This approximation Coordinate preparation with ADT. Successful docking and
reduces the size of the conformational space, which allows it to virtual screening require careful attention to the quality of the
be searched reliably, and it reduces the computational effort of coordinates used for receptors and ligands. Both AutoDock and
scoring each trial conformation. When applying these docking AutoDock Vina use a simplified representation of the molecules,
methods to a given receptor, it is important to consider the possi- which is included in a modified Protein Data Bank (PDB) file
ble effects of this limitation, and if the system includes significant format, termed PDBQT:
receptor motion a number of methods may be used, including
the following: United atom representation: both methods require coordinate
sets that include the polar hydrogen atoms. AutoDock uses polar
Using receptor structures taken from receptorligand complexes, hydrogen coordinates during docking; AutoDock Vina uses them
in which there is some expectation that the receptor is in the to assign the hydrogen bonding state of the heteroatoms, but it
relevant conformation. does not use explicit hydrogens during the docking.

906 | VOL.11 NO.5 | 2016 | nature protocols

 protocol
Atom typing: both methods require a simplified typing of small fragment molecules, such as those used in fragment-based
atoms, including identification of aromatic and aliphatic drug discovery23.
carbon atoms and identification of the hydrogen bonding state Active site prediction: docking of ligands to the entire surface of a
of heteroatoms. protein is often computationally prohibitive. The AutoDock suite
Atomic charges: AutoDock uses GasteigerMarsili atomic charges includes the AutoLigand program for identifying likely binding
for calculation of electrostatic interactions and desolvation sites on a receptor surface25. AutoLigand predicts optimal sub-
energies. AutoDock Vina does not use atomic charges. strate envelopes for binding sites on the basis of the free-energy
Ligand flexibility: both methods require the user to specify the force field of AutoDock. From coordinates for the receptor,
torsional degrees of freedom in ligand molecules and in any AutoLigand calculates and analyzes maps of interaction energy
receptor side chains that are to be flexible. to output coordinate files that show optimal envelopes for ligand
Search space: both methods require the user to specify a binding to the receptor.
docking box covering the space around the receptor that will
be searched. Limitations of the protocol
The AutoDock suite is designed to solve a specific problem: the
The graphical user interface ADT provides methods for creating docking of small, drug-like molecules to biological macromol-
suitable coordinate files from files in a variety of common ecules of known structure. It has been implemented, calibrated
formats. Automated scripts are also available for batch processing and tested with a diverse set of proteinligand complexes of bio-
2016 Macmillan Publihers Limited. All rights reserved.

of libraries of compounds. logical and medicinal interest, and it is expected to perform con-
Preparation of coordinate files (Steps 14) is arguably the most sistently with this type of system, as described in more detail in
important aspect of the process, as the quality of coordinates will the ANTICIPATED RESULTS below.
affect all results from docking. After preparation, take a crucial Systems that deviate from these design parameters will give
look at the coordinate files and examine the protonation state variable results, and they should be approached with caution.
and charges (particularly metals, if present) to see whether they Users most often encounter two significant limitations. First,
are consistent with knowledge of the system. For instance, ADT users often want to dock very large ligands, such as decapep-
does not provide any charges for metal ions; therefore, charges tides. These ligands present too many degrees of freedom, and
need to be manually added in the PDBQT file using a text editor. docking methods are not able to search the accessible confor-
Receptor coordinate files deposited in the PDB often have many mational space. Most often, the best way to solve this problem
challenges that need to be addressed. Double-check to make sure is to break it down into smaller pieces. Second, the protein targets
that you have the proper biological unit, that essential residues often show significant conformational flexibility, which is
or loops are not missing from the coordinate set, that you have not modeled in the AutoDock suite, apart from the selective
chosen only one set of coordinates in which there are alternate side-chain motion, including in this protocol. This problem is
conformations in the deposition and that you have included only typically approached through the generation of conformations
essential cofactors and structural waters. of the protein by other methods (such as molecular dynamics)
before docking.
Advanced methods. The default methods used in AutoDock
and AutoDock Vina are highly effective for typical drug-like About the protocol
ligands, and they have been widely used for applications such This protocol describes many of the steps involved in a typical
as virtual screening25. However, several refinements have been drug discovery project. The target for the protocol is the kinase
developed to approach problems that present challenges with domain of the proto-oncogene tyrosine protein kinase c-Abl.
the default methods. The protein is an important target for cancer chemotherapyin
particular, the treatment of chronic myelogenous leukemia. This
Receptor flexibility: arguably the greatest limitation in these types example was chosen because it demonstrates the use of the proto-
of docking methods is the rigid model of the receptor. AutoDock col on an important cancer target with an approved drug, Gleevec
and AutoDock Vina both allow limited flexibility of selected (imatinib), and it can show the impact of the drug-resistant muta-
receptor side chains, as described in this protocol. For systems tions of the target. The protocol covers the following:
with larger motions of loops or domains, the relaxed complex
method26 has shown success by sampling a variety of receptor Re-docking experiments using the structure of c-Abl with
conformations using molecular dynamics and then performing the anticancer drug imatinib (PDB entry 1iep (ref. 27)), using
docking simulations on these snapshots. AutoDock Vina and AutoDock.
Explicit hydration: interactions between biological molecules are Virtual screening with Raccoon2 of a library of compounds
often mediated by ordered water molecules. We have developed against c-Abl, using protein coordinates from PDB entry 1iep.
a method that uses the existing version of AutoDock but modi- Cross-docking of imatinib with c-Abl coordinates from PDB
fies the force field to model explicit water molecules. The ligand entry 1fpu (ref. 28), modeling flexibility in a threonine that
is decorated with an ensemble of water molecules, which may interacts with the drug.
or may not then contribute to the interaction based on a modi- Prediction of optimal ligands for c-Abl using AutoLigand.
fied energy evaluation grid. In tests, this hydration has shown An example of docking with explicit water molecules, which can
improvement in the prediction of bound conformations of improve results for fragment-based drug design.

nature protocols | VOL.11 NO.5 | 2016 | 907

 protocol

MATERIALS
EQUIPMENT Hardware and software
Starting data Computer: Linux, Macintosh or Windows PC; Internet access
Coordinate file for receptor (in a variety of formats, including pdb, For virtual screening with Raccoon, a Linux cluster/HPC with either a
mol2, cif and sdf) PBS or SGE scheduler
Coordinate file for ligand (in a variety of formats, including pdb, mol2, Text editor
cif and sdf) AutoDock: http://autodock.scripps.edu (information on installation is
Several files are available in Supplementary Data for use as a tutorial available at http://autodock.scripps.edu/downloads/autodock-registration/
for each of the protocols: 1iep_receptorH.pdb (coordinates of c-Abl autodock-4-2-download-page/)
kinase domain from PDB entry 1iep, with hydrogen atoms added in AutoDock Vina: http://vina.scripps.edu (information on installation is
AutoDockTools), 1iep_ligandH.pdb (coordinates of imatinib from available at http://vina.scripps.edu/manual.html#faq)
PDB entry 1iep, with hydrogen atoms added in AutoDockTools), AutoDockTools (part of MGLTools): http://mgltools.scripps.edu
1fpu_receptorH.pdbqt (coordinates of c-Abl kinase domain from (information on installation is available at http://mgltools.scripps.edu/
PDB entry 1fpu, in PDBQT format), imatinib.pdbqt (coordinates of downloads)
imatinib in PDBQT format) and NCIdivII_subset (a folder that includes AutoLigand is part of AutoDockTools
499 compounds from the ZINC library, formatted as PDBQT files) Raccoon is available at http://autodock.scripps.edu/resources/raccoon
2016 Macmillan Publihers Limited. All rights reserved.

PROCEDURE
Coordinate preparation with ADT TIMING 10 min
1| Generate the ligand coordinate file. A coordinate set that includes hydrogen atoms is required. This may be obtained in
a variety of ways, including with experimental coordinates from the PDB (http://www.rcsb.org/) or Cambridge Crystallographic
Database (http://www.ccdc.cam.ac.uk/), or via structure generation methods such as the CACTUS server (http://cactus.nci.
nih.gov/translate/). The file 1iep_ligandH.pdb is provided for use as a tutorial for this protocol (all example files are
supplied in Supplementary Data). This file includes ligand coordinates taken from PDB entry 1iep, to which all hydrogen
atoms have been added and manually adjusted to the known protonation state. Start ADT (Fig. 1) and set the working
directory by clicking File Preferences Set. Type your working directory path name into the Startup Directory box
and click Set. Click Dismiss at the bottom of the window.

2| Read the atomic coordinates. To do this, first select Ligand Input Open and use the Files of type menu to
choose PDB files. Click on your coordinate file (in this case 1iep_ligandH.pdb) and click Open. ADT will read the coordi-
nates, add charges if necessary, merge nonpolar hydrogens and assign appropriate atom types. At this point, the ligand will
be displayed in the viewer window, with aromatic carbons in green. Click OK on the popup to continue.
CRITICAL STEP If your coordinate set does not include hydrogen positions, click File Read Molecule and choose your
coordinate file. Next, click Edit Hydrogens Add to add all hydrogens by default. Select Ligand Input Choose to
choose the ligand molecule.

3| Prepare a PDBQT file by selecting Ligand TorsionTree DetectRoot; this will define the center of the torsion tree.
Selecting Ligand TorsionTree ChooseTorsions will launch a window that allows choice of torsional degrees of freedom.
Rotatable bonds are in green, rigid portions are in red and potentially rotatable bonds that are currently set as not rotatable
(such as the peptide bond at the center of imatinib) are
in magenta. Clicking on bonds will switch the rotation
flexibility on and off. When this step has been completed, PMV menus
click Done. Click Ligand Output SaveAsPDBQT, and ADT menus
then select Save to write the file 1iep_ligandH.pdbqt.
? TROUBLESHOOTING

4| Generate the receptor coordinate file. A receptor coor- Dashboard

dinate file with all hydrogen atoms is required. If you are
using experimental structures (for instance, from the PDB),

Interactive Viewer
Figure 1 | AutoDockTools (ADT). ADT is built within the Python
Molecule Viewer (PMV). ADT commands for coordinate preparation,
docking and analysis are available through menus on the lower toolbar.
PMV commands for higher-level visualization are available on the upper
tool bar. The Dashboard controls representation, colors and labeling of
molecular objects that are displayed in the Interactive Viewer panel.
For more information on the capabilities of PMV and ADT, see http://
mgltools.scripps.edu/documentation.

908 | VOL.11 NO.5 | 2016 | nature protocols

 protocol

use a text editor to remove water, ligands, cofactors, ions and so on that should not be included in the receptor.
The file 1iep_receptorH.pdb is provided for use as a tutorial for this protocol, and it includes receptor coordinates that are
taken from PDB entry 1iep. Open the file by selecting Grid Macromolecule Open, and use the Files of type menu to
choose all files. Click on your coordinate filein this case 1iep_receptorH.pdband click Open. ADT will read coordinates,
add charges, merge nonpolar hydrogens and assign appropriate atom types. Click OK to accept the changes. A window will
pop up to write the PDBQT file. Click Save to write the file 1iep_receptorH.pdbqt.
CRITICAL STEP If your coordinate set does not include hydrogen positions, Click File Read Molecule and choose your
coordinate file. Next, select Edit Hydrogens Add to add all hydrogens by default. Click Grid Macromolecule
Choose to choose the receptor molecule.
? TROUBLESHOOTING

Methods for docking simulation

5| Once receptor and ligand coordinates are formatted, the AutoDock suite provides a number of methods for docking
simulation. This protocol includes six methods, which range from a simple docking to advanced methods, as described in the
following table.
2016 Macmillan Publihers Limited. All rights reserved.

Option
(Step 5) Method Description

A Single-docking experiment with AutoDock Vina Basic docking method for study of a single ligand with a single receptor
B Single-docking experiment with AutoDock Basic docking method for study of a single ligand with a single
receptor, with explicit calculation of affinity maps
C Virtual screening with Raccoon2 and AutoDock Vina Virtual screen of a library of ligands with a single receptor, often
used for drug discovery
D AutoDock Vina with flexible side chains Docking method for a single ligand with a single receptor, incorporat-
ing limited receptor flexibility
E Active site prediction with AutoLigand Method for analysis of receptor binding sites, for prediction of
druggable sites
F Docking with explicit waters Advanced docking method for a single ligand with a single receptor
incorporating explicit bridging water molecules

(A) Single docking experiment with AutoDock Vina TIMING 30 min

(i) Generate a configuration file (Box 1) for AutoDock Vina that specifies the PDBQT files for the ligand and receptor
and defines the docking parameters. To do this, restart ADT and set the default working directory (see Step 1).
Select Grid Macromolecule Choose/Open. Choose is used when coordinates have already been read into ADT
(as in the coordinate setup above), and Open is used to read coordinates from a file. For this protocol, we are
assuming that we have restarted ADT and need to read from coordinate files. Open the receptor PDBQT file, and
click Yes to preserve the existing charges in the file and OK to accept. There may also be a warning window if there
are slight irregularities in charges; click OK if it appears.
(ii) Select Grid SetMapTypes OpenLigand, choose the ligand PDBQT file and click Open. Grid GridBox opens a
window for defining the center and size of the search space. Center CenteronLigand will define the box on the

Box 1 | Vina configuration file

receptor = 1iep_receptorH.pdbqt
ligand = 1iep_ligandH.pdbqt
center_x = 15.19
center_y = 53.903
center_z = 16.917
size_x = 15.0
size_y = 17.25
size_z = 15.0
out = 1iep_ligandH_out.pdbqt

nature protocols | VOL.11 NO.5 | 2016 | 909

 protocol

basis of the ligand. Other options are available, or the values may be changed manually with the thumbwheels. When
this step has been completed, choose File CloseSavingCurrent in the window Docking Output VinaConfig and
write the configuration file (default name: config.txt) by clicking Save.
(iii) Run AutoDock Vina. The imatinib ligand used in this protocol is challenging, and Vina will occasionally not find the
correct pose with the default parameters. Vina provides a parameter called Exhaustiveness to change the amount
of computational effort used during a docking experiment. The default exhaustiveness value is 8; increasing this to
about 24 will give a more consistent docking result. There are two ways to run AutoDock Vina: from ADT or from the
command line. From ADT, select Run RunAutoDockVina, and in the popup window use the Browse buttons to
specify the path to the AutoDock Vina executable file vina and the path to the configuration file. Edit the Cmd line
to change the exhaustiveness, such as
./vina --config config.txt --exhaustiveness=24
Click the Launch button to run the program.
Programs in the AutoDock suite may also be run at the command line. To do this, instead open a terminal window
and change to the directory that contains the coordinate files and configuration file. Next, issue the command listed
above. This command assumes that the AutoDock Vina executable vina is also located in the same directory.
2016 Macmillan Publihers Limited. All rights reserved.

Running AutoDock Vina will write a docked coordinate file 1iep_ligandH_out.pdbqt and also present docking
information to the terminal window. The predicted free energy of binding should be about 13 kcal mol1 for poses
that are similar to the crystallographic pose. With exhaustiveness set to 24, Vina will most often give a single docked
pose with this energy. With the lower default exhaustiveness, several poses flipped end to end, with less favorable
energy, may be reported.
? TROUBLESHOOTING
(iv) To visualize the results from AutoDock Vina (Fig. 2), launch ADT and set the working directory (see Step 1). Select
Analyze Dockings OpenAutoDockVinaResult and choose the output file from Step 5A(iii). Choose the default
of Single molecule with multiple conformations and click OK. This will show coordinates for each docked result.
Use the arrow keys on the keyboard to scroll through the poses. Click Analyze Macromolecule Open to choose
the receptor coordinate file to visualize the receptor 1iep_receptorH.pdbqt. Select File ReadMolecule to choose the
ligand coordinate file 1iep_ligandH.pdb, visualize the crystallographic location of the ligand and compare it with
the docked conformation.
Analyze Dockings ShowInteractions will launch a visualization that highlights interactions between the
ligand and the receptor.
(B) Single docking experiment with AutoDock TIMING 60 min
(i) Generate a grid parameter file for AutoDock that specifies the PDBQT files for the receptor and parameters for
generating the atomic affinity maps. Start ADT and set the default working directory (see Step 1). Select Ligand
Input Open and choose the ligand PDBQT file (1iep_ligandH.pdbqt). Select Grid Macromolecule Open and
choose the receptor PDBQT file (1iep_receptorH.pdbqt). Set the map types by selecting Grid SetMapTypes
ChooseLigand and choosing the ligand PDBQT file
(1iep_ligandH.pdbqt). Define the search space by
a
selecting Grid GridBox, which will open a window
for specifying the center and size of the search space.
Use Center CenteronLigand to define a minimal
box. When finished, choose File CloseSavingCurrent. AutoDock
Finally, select Output SaveGPF to save the AutoDock Vina

grid parameter file, which is typically given the

extension .gpf (use 1iep.gpf).
(ii) Run AutoGrid, either from ADT or from the command
line. From ADT, select Run RunAutoGrid, and in the
popup window use the Browse buttons to specify the
b
Thr 315

Figure 2 | Results of docking imatinib to its receptor in bound and apo

conformations. (a) Re-docking of flexible imatinib to rigid Abl (PDB
entry 1iep) using AutoDock (pink) and AutoDock Vina (green). The X-ray
crystallographic ligand position is colored by atom type. (b) Cross-docking
of flexible imatinib to Abl (PDB entry 1fpu) with a single flexible residue
side chain using AutoDock Vina (green). Note that Vina does not retain
hydrogen atom positions during docking, so the threonine hydroxyl
hydrogen is placed in a random position in the docked coordinate set.

910 | VOL.11 NO.5 | 2016 | nature protocols

 protocol

path to the AutoGrid executable file and the path to the grid parameter file. After you specify the grid parameter file,
ADT will suggest a name for the log file. Click the Launch button to run AutoGrid.
Alternatively, at the command line, open a terminal window and change to the directory that contains the coordi-
nate files and grid parameter file. Then issue the following command (this command assumes that the AutoGrid execut-
able autogrid4 is also located in the same directory):
./autogrid4 -p 1iep.gpf -l 1iep.glg
? TROUBLESHOOTING
(iii) (Optional) Visualize AutoGrid maps in ADT (see Step 5E). Start ADT and set the default working directory (see Step 1).
In the top toolbar, select Grid3D Read and choose a map file (such as 1iep_receptorH.C.map for the carbon affinity
map). In the popup control panel, click on the file in the Grid Name panel and then click on the Isocontour button;
ADT will display a histogram of map values. Change the max value to 0.0 and press return (or enter). Shift-click in
the histogram to calculate an isocontour; you can then drag the bar in the histogram right and left to change the
contour level. Values in the range of 0.5 to 0.1 are effective. The menu option File ReadMolecule can be used
to display the receptor.
(iv) Generate the docking parameter file that specifies the PDBQT file for the ligand and parameters for the
2016 Macmillan Publihers Limited. All rights reserved.

docking simulation. Select Docking Macromolecule SetRigidFilename and choose the receptor PDBQT file
(1iep_receptorH.pdbqt). Select Docking Ligand Choose, choose the ligand PDBQT file (1iep_ligandH.pdbqt)
and accept the default ligand docking parameters, which will randomize the pose of the ligand before docking.
Select Docking SearchParameters GeneticAlgorithmParameters and use the default parameters for most
drug-sized ligands, except set Number of GA Runs to 50. Additional docking parameters are specified with Docking
DockingParameters; use default parameters for most drug-sized ligands. Finally, write the docking parameter file
by selecting Docking Output LamarckianGA, which is typically given the extension .dpf (use 1iep.dpf).
(v) Run AutoDock, either from ADT or at the command line. To run from ADT, select Run RunAutoDock, and in the
popup window use the Browse buttons to specify the path to the AutoDock4 executable file and the path to
the docking parameter file. After you specify the docking parameter file, ADT will suggest a name for the log file.
Click the Launch button to run the program.
Alternatively, at the command line, open a terminal window and change to the directory that contains the
coordinate files and docking parameter file. Then issue the following command (this command assumes that the
AutoGrid executable autodock4 is also located in the same directory):
./autodock4 -p 1iep.dpf -l 1iep.dlg

? TROUBLESHOOTING
(vi) Visualize AutoDock results (Fig. 2a). Start ADT and set the default working directory (see Step 1), and to access the
results select Analyze Dockings Open and choose the 1iep.dlg file. There are several options in ADT for visual-
izing results: select Analyze Conformations
PlayRankedByEnergy and click on the arrow buttons
to scroll through conformations, or select Analyze
Docking Load to open a clickable window with
ranked conformations and energies.
? TROUBLESHOOTING
(C) Virtual screening with Raccoon2 and AutoDock
Vina TIMING 60 min
(i) Start Raccoon2 and configure the server. Raccoon
is designed to run virtual screening on a large
computational resource, such as a Linux cluster.
When you add a new server, you must configure
the connection and install one or more docking
services. Launch Raccoon2 (Fig. 3) and create a
new server connection by clicking the three-gear
icon to open the Connection Manager. Several
options need to be set: server name (a name to
identify the resource); address (the host name
or IP address of the server); and a username
and password. Click Save and close the Figure 3 | Raccoon2. Tabs at the top allow the user to choose each of the
Connection Manager. steps for setting up, running and analyzing a virtual screen.

nature protocols | VOL.11 NO.5 | 2016 | 911

 protocol
Install a docking service by selecting the server in the Connection menu. Click on the raccoon-paw icon to
prepare (raccoonize) the server. Click on the Service Manager icon (a wand) and add a new service by clicking on
the plus icon, and give it a descriptive name of your choice (Vina docking service). Click on Install AutoDock Vina
on the server, and accept several default selections. Click Save and close the Service Manager. Finally, from the
Available Services panel, select the Vina docking service.
? TROUBLESHOOTING
(ii) Set up the Ligand library. This protocol describes virtual screening of c-Abl with a small library of 500 compounds
that includes the known inhibitor imatinib. Tools for the generation and processing of ligand libraries are available
at http://autodock.scripps.edu/resources/databases.
Ligand libraries are stored on the server and made available for docking jobs, to reduce redundancy and to allow
tracing and reproducing experiments.
In the LIGANDS tab, select AddLigand ScanDirectory and browse to the directory containing the ligand library.
Close the report window and select Upload, type a descriptive library name of your choice and click Start. Close after
the upload is complete, and close the Library Manager. Right-click to select the desired ligand library.
(iii) Set up the receptor coordinates. In the RECEPTORS tab, select Add ScanDirectory, browse to the receptor PDBQT
file (created in Step 4) and click OK. Close the report window.
2016 Macmillan Publihers Limited. All rights reserved.

(iv) Configure AutoDock Vina docking parameters. In the CONFIG tab, load a receptor structure in the 3D viewer by
selecting files in the Receptor list. Use Center and Size thumbwheels to define the search space, or place the mouse
cursor over boxes and type numeric values. Modify docking parameters if desired, or load the config.txt file created
in Step 5A(i).
(v) Perform the virtual screening calculation. In the JOB
MANAGER tab, ensure that all Cluster submission
requirements are green, and then click Submit.
Several options will need to be specified: set
Project to new and type a descriptive name of
your choice; set Experiment to new and type a
descriptive name of your choice; and click OK to
start the calculation.
To follow the status of the docking, right-click
on the experiment name and select update status.
When one or more jobs in the experiment have
been completed, download them individually by
right-clicking on the job name, or download them
together by right-clicking on the experiment, and
select Download results.
(vi) Filter and analyze the results. To assist with filtering
and selection, Raccoon calculates docking pose
properties such as interaction, score and ligand
efficiency. In the ANALYSIS DATA SOURCE tab,
click on Add results Select downloaded results,
and browse to the results directory. Choose the
receptor file to be used for processing results, and
type a descriptive log file name or select the config.
txt file generated at Step 5A(i).
The filter panel allows a variety of filters to be
applied to the results (Fig. 4):
Energy will show poses within an energy range;
Ligand Efficiency will select poses within a range
of ligand efficiency; and with Target Interactions,
use + to filter by selected interactions with
receptor atoms.

Figure 4 | Raccoon result filtering. The Data Source window (top) has
several options for filtering the results of a virtual screen. Two interaction
filters are applied here. The Visualization window (bottom) allows
visualization of the filtered results, and checkboxes can be used to select
the desired compounds for export.

912 | VOL.11 NO.5 | 2016 | nature protocols

 protocol

For the c-Abl virtual screen described in this protocol, click on the + button and add an HB acceptor filter
with MET318, and then press return (note: the interface currently requires that the residue name be in all capital
letters). Click on the + button and add an HB any filter with THR315, and then press enter. Switch the menu from
match any to match all and update the filters. This will select the small number of molecules that show both of
these interactions. The THR319 position of c-Abl is known to mutate to obtain resistance. To find molecules that
do not interact with this position, click on the filter button for THR315 added above, and switch the filter from
wanted (green) to not wanted (red).
? TROUBLESHOOTING
(vii) Export results. Once a set of ligands is filtered, select interesting ones by clicking on the button in the Results panel.
Use the EXPORT tab to export the selected candidates.
(D) AutoDock Vina with flexible side chains TIMING 60 min
(i) Generate receptor coordinate files. The receptor coordinates are split into two PDBQT files: one for the rigid portion
and one for the flexible side chains. As with the rigid docking protocols, the method requires a receptor coordinate
file that includes all hydrogen atoms. This protocol describes the cross-docking of imatinib to c-Abl in PDB entry 1fpu,
treating Thr315 as flexible.
Start ADT and set the default working directory (see Step 1), select Grid Macromolecule Open and
2016 Macmillan Publihers Limited. All rights reserved.

choose 1fpu_receptorH.pdbq. Select residue(s) to be treated as flexible by selecting FlexibleResidues Input

ChooseMacromolecule, and browse to the receptor coordinate file. Several selection methods are available, including
direct selection and using the Select menu. With Select SelectFromString in the Python Molecule Viewer (PMV)
toolbar, choose Residue, type THR315 and click Add. ADT will highlight the selected residue with small yellow
crosses. Click Dismiss. Choose this selected residue to be treated as flexible with the command FlexibleResidues
ChooseTorsionsinCurrentlySelectedResidues. Finally, select FlexibleResidues Output SaveFlexiblePDBQT to save
the flexible coordinate file 1fpu_flex.pdbqt and FlexibleResidues Output SaveRigidPDBQT to save the rigid
receptor coordinate file 1fpu_rigid.pdbqt.
(ii) Generate parameter files for AutoDock Vina. To include the mobile residue, a larger grid box must be used: its bounds
can be found using the Grid GridBox tool. For this protocol, enter them manually. In ADT, select Docking
Output VinaConfig and use Browse to select the rigid (1fpu_rigid.pdbqt) and flexible (1fpu_flex.pdbqt) receptor
files. Make sure to use the 1fpu_rigid.pdbqt for the rigid file, not the full receptor PDBQT file. Click Browse to select
the ligand coordinate file 1iep_ligandH.pdbqt (Step 3), type 15.19, 53.9, 14.6 into the Center boxes and type 15.0,
17.25, 19.5 into the Size boxes. Change the Output filename to config_flex.txt, and change the Out filename (this is
the file for results) to 1fpu_ligandH_flex.pdbqt. This will create a configuration file called config_flex.txt (Box 2).
(iii) Perform flexible side-chain docking in AutoDock Vina at the command line:
./vina --config config_flex.txt --exhaustiveness 24
The exhaustiveness parameter performs additional docking simulations. The default value is 8; a value three or four
times larger works well for the current system.
(iv) Analyze the flexible docking results using ADT (Fig. 3b), as described in Step 5A(iv).
? TROUBLESHOOTING
(E) Active site prediction with AutoLigand TIMING 30 min
(i) AutoLigand identifies optimal regions for ligand binding by analyzing AutoGrid interaction energy maps for three
atom types (C, OA and HD) and the electrostatics and desolvation maps, all calculated with a 1 grid spacing.

Box 2 | AutoDock Vina configuration file for flexible docking

receptor = 1fpu_rigid.pdbqt
ligand = 1iep_ligandH.pdbqt
flex = 1fpu_flex.pdbqt
center_x = 15.19
center_y = 53.903
center_z = 14.661
size_x = 15.0
size_y = 17.25
size_z = 19.5
out = 1fpu_ligandH_flex.pdbqt

nature protocols | VOL.11 NO.5 | 2016 | 913

 protocol

The first step is to calculate the energy maps. Start ADT and set the default working directory (see Step 1). In Grid
Macromolecule Open, select a coordinate file for receptor (1iep_receptorH.pdbqt). By default, ADT will name the map
files on the basis of this file name. In Grid SetMapTypes Directly, edit the atom list to be C HD OA.
In Grid GridBox, adjust the size to cover the entire protein, and set the spacing to 1 . For 1iep, use a size of
60, 60, 70 (put the mouse cursor over the dial, type in values, and press return (or enter)), and save the parameters
using File CloseSavingCurrent. Write the grid parameter file with Grid Output Save GPF with the filename
1iep.gpf. Run AutoGrid at the command line (see Step 5B(ii)):
./autogrid4 -p 1iep.gpf -l 1iep.glg &
(ii) AutoLigand is not installed by default, and it must be loaded into ADT: select File BrowseCommands. Next, in
Select a Package, select AutoDockTools; in Select a Module, select AutoLigand Command; click Load Select Module
and then click OK. This will add an AutoLigand entry to the ADT Compute menu.
(iii) AutoLigand may be run in two modes. This protocol will generate ligand envelopes individually, by manually picking
a seed point and a volume in ADT. This mode is appropriate if you know the location of the active site of your target.
AutoLigand may also be run at the command line to scan the entire surface of the protein, predicting the location of
optimal binding sites. This mode is described in more detail on the AutoDock website.
2016 Macmillan Publihers Limited. All rights reserved.

Select File ReadMolecule and read 1iep_receptorH.pdbqt (from Step 4), and then select File ReadMolecule
and read 1iep_ligandH.pdbqt from Step 3 (the ligand coordinates are not used by AutoLigand, but they will be used
to locate the active site in this protocol). Start the AutoLigand computation with Compute AutoLigand
RunAutoLigand. This opens a window that allows you to choose the seed point and size of the envelope. Use the
sliders to place the yellow seed point marker near the ligand/active site, or shift-click on an atom in the ligand to
place the marker there. Use the default of 100 for the volume of the envelope. Start the AutoLigand calculation by
clicking OK. When AutoLigand finishes, a pop-up window will display the results. The envelope will be displayed in
the viewer (Fig. 5), and a PDB file will be written with coordinates of the envelope.
(F) Docking with explicit waters TIMING 60 min
(i) This method adds dummy atoms to the ligand
that correspond to all possible sites of hydration. a b
A modified AutoGrid map is then used during docking,
giving a favorable score when the water is well placed
and omitting the water if it overlaps with the receptor.
A final script analyzes the docked results, retaining
only those waters in appropriate positions.
This protocol assumes that the ligand and receptor
have been prepared for a standard AutoDock docking.
Two coordinate files, ligand.pdbqt and protein.pdbqt,
created using the same protocol as that described in
Steps 14 in the above example, have been provided
for this protocol, along with a modified parameter file
for the force field, a modified docking parameter file
and several Python scripts for performing each step. Carbon affinity map
To add water positions to the ligand, type at the
command line: c d
$ python wet.py -i ligand.pdbqt
This script adds the dummy W atoms to the ligand
PDBQT file, saving it to the file ligand_HYDRO.pdbqt.
(ii) Calculate the default atomic grid maps. Start ADT
and set the default working directory (see Step 1).
Select Ligand Input Open and choose the

Figure 5 | AutoLigand results. c-Abl is analyzed with AutoLigand.

(a) c-Abl with imatinib. (b) Carbon affinity map calculated around the
protein. Notice the many disconnected areas of strong affinity. (c) 100-point
envelope identifies the regions of the active site that provide the strongest
affinity for the drug. (d) 400-point envelope includes the region of high
affinity and also extends into adjacent solvent-accessible regions on 100-point envelope 400-point envelope
the protein surface.

914 | VOL.11 NO.5 | 2016 | nature protocols

 protocol

ligand PDBQT file (ligand.pdbqt), and select Grid Macromolecule Open and choose the receptor PDBQT file
(receptor.pdbqt). Set the map types in Grid SetMapTypes Directly and ensure that the HD and OA types are in
the list. Use Grid GridBox to specify the center and size of the search space. In the popup window, use Center
CenteronLigand to define a minimal box. When finished, save the parameters using File CloseSavingCurrent. Write
the grid parameter file using Output SaveGPF, with the name protein.gpf. Run AutoGrid (see Step 5B(ii) for more
information) with Run RunAutoGrid or at the command line with
./autogrid4 -p protein.gpf -l protein.glg.
(iii) Generate the W map. If standard filenames are used for the maps, only the receptor name must be specified for the
script that generates the map for the water energy evaluation:
$ python mapwater.py -r protein.pdbqt -s protein.W.map

Box 3 | Modified docking parameter file for docking with explicit waters
autodock_parameter_version 4.2 #version control
2016 Macmillan Publihers Limited. All rights reserved.

parameter_file AD4_water_forcefield.dat #***CUSTOM PARAMETER FILE

outlev 1 #diagnostic output level
intelec #calculate internal estat
seed pid time #seeds for random generator
ligand_types A C HD N NA W #***atom types in ligand, add W
fld protein.maps.fld #grid_data_file
map protein.A.map #atom-specific affinity map
map protein.C.map #atom-specific affinity map
map protein.HD.map #atom-specific affinity map
map protein.N.map #atom-specific affinity map
map protein.NA.map #atom-specific affinity map
map protein.W.map #***W-specific affinity map
elecmap protein.e.map #electrostatics map
desolvmap protein.d.map #desolvation map
move ligand_HYDRO.pdbqt #***small molecule, with W atoms
about 65.7163 39.7789 -2.5985 #small molecule center
tran0 random #initial coordinates/A or random
axisangle0 random #initial orientation
dihe0 random #initial dihedrals
rmstol 2.0 #cluster_tolerance/A
rmsref ligand_xray_HYDRO.pdbqt #reference ligand conformation
ga_pop_size 150 #individuals in population
ga_num_evals 2500000 #maximum energy evaluations
ga_num_generations 27000 #maximum number of generations
ga_elitism 1 #set elitism level
ga_mutation_rate 0.02 #rate of gene mutation
ga_crossover_rate 0.8 #rate of crossover
ga_window_size 10 #
ga_cauchy_alpha 0.0 #Cauchy alpha parameter
ga_cauchy_beta 1.0 #Cauchy beta parameter
set_ga #set the above parameters
sw_max_its 300 #Solis & Wets iterations
sw_max_succ 4 #rho parameter
sw_max_fail 4 #rho parameter
sw_rho 1.0 #size of local search space
sw_lb_rho 0.01 #lower bound on rho
ls_search_freq 0.06 #local search probability
set_psw1 #set pseudo-Solis & Wets
unbound_model bound #state of unbound ligand
ga_run 10 #do this many hybrid GA-LS runs
analysis #ranked cluster analysis

nature protocols | VOL.11 NO.5 | 2016 | 915

 protocol

(iv) Create a modified docking parameter file. Select Docking Macromolecule SetRigidFilename and choose the
receptor PDBQT file (protein.pdbqt). Select Docking Ligand Choose and choose the ligand PDBQT file
(ligand_HYDRO.pdbqt), and accept the default ligand docking parameters, which will randomize the pose of the
ligand before docking. Set the search parameters in Docking SearchParameters GeneticAlgorithmParameters,
changing Number of GA Runs to 50, and use defaults on the remaining parameters. In Docking DockingParameters,
use default parameters for most drug-sized ligands.
Write the docking parameter file using Docking Output LamarckianGA, with the name ligand_HYDRO_protein.dpf.
The standard docking parameter file must be modified manually in two ways: parameter_file AD4_water_forcefield.
dat must be added near the top and protein.W.map must be added to the list of maps. Both of these changes are
highlighted in Box 3.
? TROUBLESHOOTING
(v) Run AutoDock (see Step 5B(v) for more information) in ADT with Run RunAutoDock or at the command line with
./autodock4 -p ligand_HYDRO_protein.dpf -l ligand_HYDRO_protein.dlg.
(vi) Extract and score the results at the command line with
2016 Macmillan Publihers Limited. All rights reserved.

$ python dry.py -c -r protein.pdbqt -m protein.W.map -i ligand_HYDRO_protein.dlg

This script will filter the docking results using the receptor to identify displaced water and the W map to rank
the conserved ones as strong or weak. This will write a file called ligand_LELC_DRY_SCORED.pdbqt with the
calculated energy.

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

Table 1 | Troubleshooting table.

Step Problem Possible reason Solution

3 Incorrect protonation ADT currently uses Babel to add hydrogen Double-check the PDBQT file and manually curate
of the ligand positions, which makes occasional errors errors in protonation

4 Missing coordinates Experimental coordinate sets are heterogeneous. Double-check the PDBQT file and manually
in the receptor Common problems include missing side chains curate to include the proper biological unit.
and loops, asymmetric units that include In general, use the biological unit and omit
only a portion of the biological unit, alternate ligands and waters except for strongly bound
conformations, essential cofactors and structural prosthetic groups
waters

5A(iii) Vina does not run ADT Start Vina GUI or command line is not Locate the Vina executable on your computer,
using the proper path to the Vina executable, and use the appropriate path name. Refer
or file is incorrect type for the CPU or operating to online installation notes to locate the
system executable

No low-energy Grid box does not enclose the active site Choose an appropriate location for the grid
conformations are found box in ADT

5B(ii) AutoGrid does not run ADT Run AutoGrid GUI or command line is not Locate the AutoGrid executable on your
using the proper path name to the AutoGrid4 computer, and use the appropriate path name.
executable, or file is incorrect type for the CPU Refer to online installation notes to locate the
or operating system executable

5B(v) AutoDock does not run ADT Run AutoDock GUI or command line is not Locate the AutoDock executable on your
using the proper path name to the AutoDock4 computer, and use the appropriate path name.
executable, or file is incorrect type for the CPU Refer to online installation notes to locate the
or operating system executable

5B(vi) Re-docking experiments System has too many degrees of freedom for Use longer search protocols, or simplify the
do not reproduce the AutoDock conformational search system by reducing degrees of freedom (docking
known pose in parts or rigidifying part of the ligand)
(continued)

916 | VOL.11 NO.5 | 2016 | nature protocols

 protocol
Table 1 | Troubleshooting table (continued).

Step Problem Possible reason Solution

5C(i) Problems with selection Right-click is used in several places in Use System Preferences on Macintosh
Raccoon for selection (control-click on computers to enable right-clicking for the
Macintosh computers) mouse or trackpad

5C(vi) No results selected Residue names are case sensitive Use all capitals, such as THR315
during filtering by
interaction

5D(iv) No low-energy Grid box does not enclose all of the side chains Choose a larger grid box that encloses the
conformations are found binding side and all of the flexible side chains

5F(iv) Error message about Water dummy atom parameters not defined Ensure that the modified files are used
missing W parameters in force field parameter file and/or docking
parameter files
2016 Macmillan Publihers Limited. All rights reserved.

TIMING
The indicated timing of each step is a rough estimatethe actual times will depend on the complexity of the system being
docked and the equipment being used for the computation.
Steps 14, coordinate preparation with ADT: 10 min
Step 5A, docking with AutoDock Vina: 30 min
Step 5B, docking with AutoDock: 60 min
Step 5C, virtual screening with Raccoon2 and AutoDock Vina: 60 min
Step 5D, flexible side-chain docking with AutoDock Vina: 60 min
Step 5E, active site prediction with AutoLigand: 30 min
Step 5F, docking with explicit hydration: 60 min

ANTICIPATED RESULTS
AutoDock Vina (Step 5A) will provide coordinates for one or more optimized poses for the ligand (Fig. 2a). In our tests
of the docking of imatinib with c-Abl, we found that the default docking parameters are sufficient to give a consistent
solution in most cases. The conformational flexibility of this system is at the limit of the default docking protocol,
which may be indicated by a docking result with multiple less favorable poses. For challenging systems with high degrees
of conformational flexibility, the exhaustiveness parameter can be used to perform additional docking simulations, often
giving more consistent results. This is described in Step 5A(iii). When analyzing results from AutoDock Vina, note that
it uses the input hydrogen positions to assign hydrogen-bonding types to heteroatoms, but it does not optimize them
during docking simulation, so the hydrogen positions in the output pose are in random conformations.
The number of evaluations used in AutoDock will determine the exhaustiveness of the conformational search
(Step 5B). For ligands with 14 torsional degrees of freedom, short (250,000) or medium (2,500,000) lengths should
provide adequate searching, but for larger ligands, such as imatinib, used in this protocol, a longer search is needed,
with 10,000,00025,000,000 evaluations. The clustering analysis is the best way to determine whether the simulation
has adequately searched the available conformation space: perform multiple docking simulations and confirm that the
best conformation is found multiple times.
Virtual screening with Raccoon2 allows the docking and ranking of tens of thousands of compounds to a macromolecular
target (Step 5C). Analysis and filtering is one of the most challenging aspects of virtual screening, as the goal is to identify
a few promising leads from a large body of docked results. We have obtained effective results by filtering on the basis of
predicted docking energy combined with the presence of key interactions in the system. The addition of this type of expert
knowledge greatly improves the success rate when compounds are tested.
In our hands, roughly half of the systems we have used for docking simulations will give useful results using the default
rigid model for the receptor. In other cases, protein motion will cause problems for the prediction of reasonable poses.
AutoDock and AutoDock Vina may be configured to dock ligands with selected receptor residues treated explicitly as flexible
(Step 5D). Docking with flexible side chains is difficult and often requires additional computational effort. The test system
included with this protocol is an example. PDB entry 1fpu includes a structure of Abl with an inhibitor similar to imatinib.
Threonine 315 in the structure is flipped relative to that in 1iep, and it is unable to form a hydrogen bond with the drug.
The protocol performs a cross-docking experiment of imatinib docking to Abl from 1fpu, treating Thr315 as flexible. For
best results, AutoDock Vina needs to be run with a more exhaustive search to find the proper pose. For systems with larger

nature protocols | VOL.11 NO.5 | 2016 | 917

 protocol
Figure 6 | Docking with explicit hydration. (a) Crystallographic structure of a b c
acetylcholine-binding protein with nicotine. An ordered water molecule (red
sphere) mediates interaction with the protein. (b) The default protocol in
AutoDock finds two conformations of the pyridine ring with equal predicted
energies. (c) Hydrated docking predicts the observed conformation. The
second water molecule, marked with an X, is included during the docking but
is removed because it clashes with the protein.

motion of loops or domains, separate docking simulations may be run for different conformations of the protein, obtained
from multiple experimental structures or dynamics simulations of individual structures.
We have found explicit hydration (Step 5F) to be particularly useful for the docking of small ligands and fragment
molecules, as water-mediated interactions may form a larger portion of their interactions than for larger ligands.
In the example included here (Fig. 6), the pyridine ring on nicotine forms a water-mediated interaction with the
acetylcholine-binding protein. When the default methods in AutoDock are used, the ligand is placed in the proper position,
but two conformations are found with very similar predicted association energies, with the pyridine in the observed
position in one and rotated by 180 in the other. In the hydrated docking experiment, two water dummy atoms are placed
2016 Macmillan Publihers Limited. All rights reserved.

on the ligand: one interacting with the pyridine and the other interacting with a tertiary amino group. The best pose
places the pyridine and associated water in the experimentally observed position and deletes the other water molecule,
as it clashes with the protein.
AutoLigand analyzes the atomic affinity maps to predict the optimal locations for substrate binding (Step 5E).
The computation time is dependent on the size of the envelope, and typical drug-sized molecules fall in the range
of 50500 3 grid points. The method combines information from three mapscarbon, oxygen and hydrogento predict
the optimal atom type at each point. The oxygen map also represents likely locations for nitrogen atoms that accept
hydrogen bonds, as the force field parameters for nitrogen atom types are similar to those for oxygen. We have used
AutoLigand as a tool to identify the regions of a given ligand that are providing the most affinity, and to identify locations
on lead molecules that would provide the most additional affinity during functionalization.

Note: Any Supplementary Information and Source Data files are available in the 8. Goodsell, D.S., Lauble, H., Stout, C.D. & Olson, A.J. Automated docking
online version of the paper. in crystallography: analysis of the substrates of aconitase. Proteins 17,
110 (1993).
Acknowledgments This work was supported by the US National Institutes 9. Goodsell, D.S., Morris, G.M. & Olson, A.J. Automated docking
of Health (grant R01 GM069832 to A.J.O.). This is manuscript number 29118 of flexible ligands: applications of AutoDock. J. Mol. Recognit. 9,
from the Scripps Research Institute. 15 (1996).
10. Soares, T.A., Goodsell, D.S., Briggs, J.M., Ferreira, R. & Olson, A.J.
AUTHOR CONTRIBUTIONS All authors contributed equally to this work. Docking of 4-oxalocrotonate tautomerase substrates: implications for the
D.S.G. and S.F. authored the protocol manuscript with extensive input from the catalytic mechanism. Biolpolymers 50, 319328 (1999).
other authors, based on tutorials developed by all authors. All authors have been 11. Rosenfeld, R.J. et al. Automated docking of ligands to an artificial
instrumental in development of the AutoDock suite and training of users. active site: augmenting crystallographic analysis with computer modeling.
J. Comput. Aided Mol. Des. 17, 525536 (2003).
COMPETING FINANCIAL INTERESTS The authors declare no competing financial 12. Brik, A. et al. Rapid diversity-oriented synthesis in microtiter plates
interests. for in situ screening of HIV protease inhibitors. ChemBioChem 4,
12461248 (2003).
Reprints and permissions information is available online at http://www.nature. 13. Brik, A. et al. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis
com/reprints/index.html. of HIV-1 protease inhibitors. ChemBioChem 6, 11671169 (2005).
14. Perryman, A.L. et al. Fragment-based screen against HIV protease.
1. Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy Chem. Biol. Drug Des. 75, 257268 (2010).
of docking with a new scoring function, efficient optimization, and 15. Cosconati, S. et al. Structure-based virtual screening and biological
multithreading. J. Comput. Chem. 31, 455461 (2010). evaluation of Mycobacterium tuberculosis adenosine 5-phosphosulfate
2. Goodsell, D.S. & Olson, A.J. Automated docking of substrates to proteins reductase inhibitors. J. Med. Chem. 51, 66276630 (2008).
by simulated annealing. Proteins 8, 195202 (1990). 16. Perryman, A.L. et al. A virtual screen discovers novel, fragment-sized
3. Morris, G.M. et al. Automated docking using a Lamarckian genetic inhibitors of Mycobacterium tuberculosis InhA. J. Chem. Inf. Model. 55,
algorithm and an empirical binding free energy function. J. Comput. 645659 (2015).
Chem. 19, 16391662 (1998). 17. Cosconati, S. et al. Identification of novel beta-secretase inhibitors
4. Forli, S. Raccoon for Processing Virtual Screening <http://autodock.scripps. through inclusion of protein flexibility in virtual screening calculations.
edu/resources/raccoon> (2013). FASEB J. 22, 791798 (2008).
5. Morris, G.M. et al. AutoDock4 and AutoDockTools4: automated docking 18. Cosconati, S. et al. Protein flexibility in virtual screening: the BACE-1
with selective receptor flexibility. J. Comput. Chem. 30, 27852791 case study. J. Chem. Inf. Model. 52, 26972704 (2012).
(2009). 19. Jiang, H. et al. Stabilizers of the Max homodimer identified in virtual
6. Harris, R., Olson, A.J. & Goodsell, D.S. Automated prediction of ligand screening inhibit Myc function. Mol. Pharmacol. 76, 491502
ligand-binding sites in proteins. Proteins 70, 15061517 (2008). (2009).
7. Sousa, S.F. et al. Protein-ligand docking in the new millenniuma 20. Leonard, S.E., Garcia, F.J., Goodsell, D.S. & Carroll, K.S. Redox-based
retrospective of 10 years in the field. Curr. Med. Chem. 20, 22962314 probes for protein tyrosine phosphatases. Angew. Chem. Int. Ed. Engl. 50,
(2013). 44234427 (2011).

918 | VOL.11 NO.5 | 2016 | nature protocols

 protocol
21. Huey, R., Goodsell, D.S., Morris, G.M. & Olson, A.J. Grid-based hydrogen bond 25. Cosconati, S. et al. Virtual screening with AutoDock: theory and practice.
potentials in improved directionality. Lett. Drug Des. Discov. 1, 178183 (2004). Expert Opin. Drug Discov. 5, 597607 (2010).
22. Huey, R., Morris, G.M., Olson, A.J. & Goodsell, D.S. A semiempirical free 26. Lin, J.H., Perryman, A.L., Schames, J.R. & McCammon, J.A.
energy force field with charge-based desolvation. J. Comput. Chem. 28, The relaxed complex method: accommodating receptor flexibility
11451152 (2006). for drug design with an improved scoring scheme. Biopolymers 68,
23. Forli, S. & Olson, A.J. A force field with discrete displaceable waters 4762 (2003).
and desolvation entropy for hydrated ligand docking. J. Med. Chem. 55, 27. Nagar, B. et al. Crystal structures of the kinase domain of c-Abl in
623638 (2012). complex with the small molecule inhibitors PD173955 and imatinib
24. Bianco, G., Forli, S., Goodsell, D.S. & Olson, A.J. Covalent docking using (STI-571). Cancer Res. 62, 42364243 (2002).
autodock: two-point attractor and flexible side chain methods. Protein Sci. 28. Schindler, T. et al. Structural mechanism for STI-571 inhibition of abelson
25, 295301 (2016). tyrosine kinase. Science 289, 19381942 (2000).
2016 Macmillan Publihers Limited. All rights reserved.

nature protocols | VOL.11 NO.5 | 2016 | 919