Software News and Updates

AutoDock4 and AutoDockTools4: Automated Docking

with Selective Receptor Flexibility
GARRETT M. MORRIS,1 RUTH HUEY,1 WILLIAM LINDSTROM,1 MICHEL F. SANNER,1
RICHARD K. BELEW,2 DAVID S. GOODSELL,1 ARTHUR J. OLSON1
1

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
2
Department of Cognitive Science, University of California, San Diego, La Jolla,
California 92093
Received 10 December 2008; Revised 27 January 2009; Accepted 3 February 2009
DOI 10.1002/jcc.21256
Published online 27 April 2009 in Wiley InterScience (www.interscience.wiley.com).

Abstract: We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited exibility in the receptor. Several tests are reported here, including a
redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using exible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a
grid-based docking method and a modication of the exible sidechain technique.
q 2009 Wiley Periodicals, Inc.

J Comput Chem 30: 27852791, 2009

Key words: AutoDock; computational docking; protein exibility; covalent ligands; computer-aided drug design

Introduction
Automated docking is widely used for the prediction of biomolecular complexes in structure/function analysis and in molecular
design. Dozens of effective methods are available, incorporating
different trade-offs in molecular representation, energy evaluation, and conformational sampling to provide predictions with a
reasonable computational effort.18 AutoDock combines an empirical free energy force eld with a Lamarckian Genetic Algorithm, providing fast prediction of bound conformations with
predicted free energies of association.9
In our hands, AutoDock3 has proven to be effective in
roughly half of the complexes that we have studied. The remaining half show signicant motion of the receptor upon binding,
and thus have required a more sophisticated model of motion in
the receptor, typically performed outside of AutoDock3. The
new version of AutoDock described hereAutoDock4incorporates explicit conformational modeling of specied sidechains
in the receptor to address this problem. This capability also provides an effective method for analysis of covalently attached
ligands.

Methods

ular targets.9,1114 To allow searching of the large conformational space available to a ligand around a protein, AutoDock
uses a grid-based method to allow rapid evaluation of the binding energy of trial conformations. In this method, the target protein is embedded in a grid. Then, a probe atom is sequentially
placed at each grid point, the interaction energy between the
probe and the target is computed, and the value is stored in the
grid. This grid of energies may then be used as a lookup table
during the docking simulation.
The primary method for conformational searching is a
Lamarckian genetic algorithm, described fully in Morris et al.9
A population of trial conformations is created, and then in successive generations these individuals mutate, exchange conformational parameters, and compete in a manner analogous to biological evolution, ultimately selecting individuals with lowest
binding energy. The Lamarckian aspect is an added feature
that allows individual conformations to search their local conformational space, nding local minima, and then pass this information to later generations. A simulated annealing search
method and a traditional genetic algorithm search method are
also available in AutoDock4.
AutoDock4 uses a semiempirical free energy force eld to
predict binding free energies of small molecules to macromolec-

Overview of AutoDock4

Since its release in 1990,10 AutoDock has proven to be an effective tool capable of quickly and accurately predicting bound
conformations and binding energies of ligands with macromolec-

Correspondence to: A. J. Olson; e-mail: [email protected]

Contract/grant sponsor: National Institutes of Health; contract/grant
number: RO1 GM069832

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ular targets. Development and testing of the force eld has been
described elsewhere.11 The force eld is based on a comprehensive thermodynamic model that allows incorporation of intramolecular energies into the predicted free energy of binding. This
is performed by evaluating energies for both the bound and
unbound states. It also incorporates a new charge-based desolvation method that uses a typical set of atom types and charges.
The method has been calibrated on a set of 188 diverse protein
ligand complexes of known structure and binding energy, showing a standard error of about 23 kcal/mol in prediction of binding free energy in cross-validation studies.
Receptor Flexibility

AutoDock4 allows fully exible modeling of specic portions of

the protein, in a similar manner as the ligand. The user selects
specic sidechains that are separated from the protein. During
the simulation, these are treated explicitly, allowing rotation
around torsional degrees of freedom using the same methods
used to explore the conformational space of the exible ligand.
The remaining portion of the protein is represented using the afnity grids described earlier.
Methods for Covalent Docking

We have developed and tested two methods for docking of

covalently-attached complexes: a grid-based approach and a
modication of the exible sidechain method. The grid-based
approach calculates a special map for the site of attachment of
the covalent ligand. A Gaussian function is constructed with
zero energy at the site of attachment and steep energetic penalties at surrounding areas. The docking analysis is then performed
by assigning a special atom type in the ligand for the atom that
forms the covalent linkage. The docking simulation places this
atom within the Gaussian well. One caveat is that this approach
does not constrain the geometry of the covalent attachment to
reasonable bond angles. To overcome this limitation, we tested
the method using two Gaussian grids to dene the bond that is
formed during covalent linkage. Note, however, that the conformational freedom allowed with a single Gaussian grid may be
an advantage if the method is used, for instance, to target
ligands to metal coordination sites.
We also tested use of the exible sidechain method for docking of covalent ligands. In this case, a coordinate le is created
with the ligand attached to the proper sidechain in the protein,
by overlapping ideal coordinates of the ligand onto the proper
bond in the protein. This sidechain-ligand structure is then
treated as exible during the docking simulation, searching torsional degrees of freedom to optimize the interaction with the
rest of the protein.
Overview of AutoDockTools

With the release of AutoDock3, it became apparent that the

tasks of coordinate preparation, experiment design, and analysis
required an effective graphical user interface to make AutoDock
a widely accessible tool. AutoDockTools was created to ll this
need. AutoDockTools facilitates formatting input molecule les,
with a set of methods that guide the user through protonation,

calculating charges, and specifying rotatable bonds in the ligand

and the protein (described later). To simplify the design and
preparation of docking experiments, it allows the user to identify
the active site and determine visually the volume of space
searched in the docking simulation. Other methods assist the
user in specifying search parameters and launching docking calculations. Finally, AutoDockTools includes a variety of novel
methods for clustering, displaying, and analyzing the results of
docking experiments.
AutoDockTools is implemented in the object-oriented programming language Python and is built from reusable software
components.15,16 The easy-to-use graphical user interface has a
gentle learning curve and an effective self-taught tutorial is
available online. Reusable software components are used to represent the exible ligand, the sets of parameters and the docking
calculation, enabling a range of uses from a single use to thousands of docking experiments involving many different sets of
molecules, facilitating automated high-throughput applications.
For example, converting the NCI diversity database of small
molecules into AutoDock-formatted ligand les was possible
with a short Python script of less than 20 lines by leveraging the
existing software components underlying AutoDockTools.
AutoDockTools exists in the context of a rich set of tools for
molecular modeling, the Python Molecular Viewer (PMV).16,17
PMV is a freely distributed Python-based molecular viewer. It is
built with a component-based architecture with the following
software components: ViewerFramework, a platform for interactive visualization; Deja Vu, a generic OpenGL-based threedimensional viewing component; and MolKit, a hierarchical data
representation of molecules. AutoDockTools consist of a set of
commands dynamically extending PMV with commands specic
to the preparation, launching and analysis of AutoDock calculations. Hence, all PMV commands (such as reading/writing les,
calculating and displaying secondary structure, adding or deleting
hydrogens, calculating charges and molecular surfaces, and many
others) are also naturally available in AutoDockTools. PMV also
provides access to the Python-interpreter so that commands or
scripts can be called interactively. PMV commands log themselves, producing a session le that can be rerun. In summary,
AutoDockTools is an example of a specialization of the generic
molecular viewer PMV for the specic application of AutoDock.
Specication of Ligand and Receptor Flexibility
in AutoDockTools

AutoDockTools provides an interactive method for dening the

torsional tree for a given ligand and receptor. Each step in this
process has been automated to allow automatic assignment, for
use in batch processes such as virtual screening. Ligand exibility is assigned in several steps. First, a root atom is chosen,
which will act as the xed position during coordinate transformation in the docking simulation. To nd the optimal atom, we
evaluate the number of atoms in each branch, and choose the
root atom that minimizes the size of the largest branch. In some
cases, the user may wish to limit the exibility of the ligand.
AutoDockTools provides two choices to do this assignment
automatically. One choice selects the set of torsional degrees of
freedom that will move the largest number of atoms (torsions

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AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility

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Figure 1. Redocking results. Results of redocking of 188 diverse ligand-protein complexes. Open
squares represent complexes where the docked conformation with best predicted energy was less that
RMSD relative to the experimentally observed conformation. Dots are complexes where the best
3.5 A
RMSD from the experimental structure.
docked conformation was greater than 3.5 A

near the root), the other adds torsions progressively from the
leaves, moving the fewest number of atoms and leaving the core
of the molecule rigid.
Validation Data Sets

Two sets of complexes were used for the validation of AutoDock4, both of which have been described previously.11 A set
of 188 diverse protein-ligand complexes were taken from the
Ligand-Protein Database (http://lpdb.scripps.edu) and a set of 87
HIV protease complexes where taken from the PDBBind database (http://www.pdbbind.org). All coordinates were checked
manually for the proper biological unit and consistency in naming schemes. Hydrogen atoms and charges were added in AutoDockTools, using Babel for hydrogens and the Gasteiger
PEOE18 method for charges. Several misassigned charges were
modied manually, as described in the previous report.
The 87 HIV protease complexes were aligned to allow easy
comparison of docked conformations during cross dockings. An
analysis of steric clashes was performed by swapping ligands
within the set of 87 aligned complexes. ARG8 showed the largest number of bad contacts, with 877 cases where a ligand atom
distance. Other
contacted a sidechain atom with less than 2 A
contacts occurred with ILE50 (140 cases), PRO81 (95 cases),
and PHE82 (88 cases, in two V82F mutants).
Docking Experiments

Docking experiments were performed with AutoDock4 and compared with docking experiments with AutoDock3. For each complex, 50 docking experiments were performed using the
Lamarckian genetic algorithm with the default parameters from
AutoDock3. A maximum of 25 million energy evaluations was

applied for each experiment. The results were clustered using a

.
tolerance of 2.0 A
In the HIV cross dockings, ligand exibility was limited to
10 torsional degrees of freedom, selecting torsions that allowed
the fewest number of atoms to move (freezing the core of the
molecule). Flexible docking was performed allowing three torsions to rotate in residue ARG8, in both the A and B chains.
The structural water (water 301) was included in complexes that
included this water in the crystallographic structure, and hydrogen atoms were added in geometry that allowed hydrogen bonding to the aps.
We have also changed the default model for the unbound
system in the current version of AutoDock. Our previous method
calculated internal energies for an extended form of the molecule, mimicking a conformation that might be expected when
fully solvated.11 Results from beta testers, however, showed that
this protocol has severe limitations when used for virtual screening. In cases where the ligand is sterically crowded, the articial
force eld used to drive the ligand into an extended conformation tends to lead to conformations with sub-optimal energy.
When the difference is calculated between this unbound conformation and the bound conformation, it leads to articially favorable predictions of the free energy of binding. In response to
this problem, we have returned to the default model of assuming
that the unbound conformation of the ligand is the same as the
bound conformation. Other options in AutoDock allow the user
to use an energy-minimized conformation of the ligand as the
unbound model.
Implementation and Availability

AutoDock 4.2 is currently being distributed free of charge

as open source under a GPL license at the WWW site: http://

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autodock.scripps.edu. ADT is being distributed free of charge as

part of the MGLTools package, at the WWW site: http://
mgltools.scripps.edu/downloads.

cessfully docked complexes with about 10 or fewer torsional

degrees of freedom, and in 97 of 188 complexes, the lowest
(data not
energy conformation showed RMSD less than 3.5 A
shown).

Results and Discussion

Redocking Analysis

Cross Docking Analysis with Protein Flexibility

Our rst test of AutoDock4 is a redocking experiment using a

set of 188 diverse protein-ligand complexes. The results, presented in Figure 1, are similar to the redocking study performed
during the energy function calibration.11 AutoDock4 successfully
redocks most complexes with about 10 or fewer torsional
degrees of freedom, but fails for most complexes with higher
conformational exibility. In 100 of 188 complexes, the docked
RMSD of
conformation with lowest energy was within 3.5 A
the crystallographic conformation. This is slightly better than a
similar redocking experiment with AutoDock3. AutoDock3 suc-

To validate the use of exible sidechains in docking, we have

used a set of 87 retroviral proteases with inhibitors. These proteins have a tunnel-shaped active site that wraps around a peptidomimetic inhibitor. An arginine-aspartate salt bridge forms at
each end of the tunnel, bridging the two subunits. In complexes
with large inhibitors, these arginines move to make space,
whereas they adopt a more closed position in complexes with
small inhibitors. In previous work, we have demonstrated that
steric clash with these arginines prevents the docking of large
inhibitors to proteins in the more closed conformation.12

Figure 2. Cross docking results. (A) Difference in energy between rigid docking and exible docking.
White points are docking experiments where the exible docking showed 20 kcal/mol more favorable
predicted free energy of docking and black points are docking experiments where the rigid docking
showed similar or worse free energy of docking. Inhibitors are ordered from small to large, with the
cyclic urea inhibitors separated at the bottom. The protease structures are ordered similarly. (B) Cross
docking with rigid protease structures. Each point is colored by the RMSD of ligand atoms from the
in white and RMSD [5 A
in black. (C) Cross docking
crystallographic structure, with RMSD 5 0 A
with ARG8 treated as exible in the protease. Each point is colored with the same scale as in (B).

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Figure 2. (Continued)

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G. M. Morris et al. Vol. 30, No. 16 Journal of Computational Chemistry

Figure 3. Results of covalent docking. (A) Using a Gaussian map centered on serine OG. The crystallographic structure is shown in large bonds and the best docked conformation is shown in thinner
bonds. The blue sphere surrounds the region of most favorable energy in the Gaussian map. (B) Using
a Gaussian map centered on serine CB. (C) Using two Gaussian maps. (D) Using a exible sidechain
to model the covalent ligand.

Using a distributed computing environment, we performed a

large cross docking experiment, taking inhibitors from 87 crystallographic structures and docking them to the protein conformations from these structures. We performed two parallel
experiments, one with a rigid protein target, and one modeling
the two ARG8 amino acids as exible. Results are shown in
Figure 2. Parameters were chosen that correspond to a typical
computational effort for an individual docking experiment, with
25,000,000 evaluations and 50 docked conformations. The torsional degrees of freedom in the ligand were limited to 10, with
an added six degrees of freedom for the two arginine sidechains
in the exible sidechain tests.
The complexes in Figure 2 are arranged to highlight the
structural features of the complexes. They are arranged from top
to bottom based on the size of the inhibitor, with small inhibitors at the top and large inhibitors at the bottom. The cyclic
urea inhibitors and similar inhibitors that are designed to displace the structural water 301 are separated at the bottom, also
ordered from small to large. The protease structures are arranged
in identical order from left to right, so the diagonal corresponds
to redocking experiments. Note that the large block at lower left
corresponds to cyclic urea inhibitors docked to protease structures that include water 301, so we dont expect that they will
dock correctly. The large block at upper right includes peptidomimetic inhibitors docked to protease structure that do not

include water 301, so we might expect some loss of specicity

in docking, although this is not observed.
As seen in Figure 2A, exible docking improves docking in
the complexes that are most expected to benet. The white band
below center corresponds to large inhibitors binding to protease
structures that were solved with small inhibitors. These complexes show the most favorable change in docking energy with
exible docking, as unfavorable contacts with the ARG8 residues are relaxed.
Looking to the RMSD results in Figure 2B, we see that rigid
docking fails with several types of complexes. The large block
of cyclic urea inhibitors at lower left show poor RMSD values,
as expected. Roughly 2/3 of the small inhibitors were docked
successfully, and the mid-size ones were very successful. The
horizontal block just below center (inhibitors 1hvk through 1hvj)
are cases where large inhibitors are docked to active sites with
ARG8 in a nonpermissive position, so these are the complexes
where we expect improvement with a exible protease model.
These results underscore the fact that in this experiment, the
larger inhibitors are actually an easier problem, for two reasons.
First, the space searched in each experiment includes the active
site and a small region outside the protein. In some cases, small
ligands nd incorrect conformations entirely outside the active
site, but the larger ligands are forced to be at least partially
inside the active site, due to their size. Second, several torsional

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AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility

degrees of freedom are frozen in the proper orientation in the

large inhibitors, and this tends to favor the crystallographic conformation.
The results for the cyclic urea inhibitors follow a similar
trend. The block at lower left are results for docking cyclic urea
inhibitors into protease structures that include the structural
water, so it is not surprising that the experiments do not nd the
proper answer. The block at lower right shows docking of cyclic
urea inhibitors with protease structures without the structural
water. It shows a similar trend of large inhibitors docking more
consistently than the smaller inhibitors. The block at upper right
shows docking of other inhibitors to proteases without the structural water. These experiments perform similarly to the experiments with the structural water.
When we add exibility to the two ARG8 at the ends of the
active site tunnel, we achieve the desired result with the large
inhibitors that contact this residue. AutoDock4 successfully
docks the large inhibitors in cases where the docking failed with
rigid receptors (Fig. 2C). Unfortunately, the experiments with
exible sidechains showed signicantly worse results for the
small inhibitors, failing in over half of the cases. This underscores another limitation of use of exible sidechains. They
increase the size of the conformational space to be searched-in
these cases, increasing it from a problem that is accessible to
AutoDock to a problem with too many degrees of freedom.
Covalent Docking

We tested two methods for docking covalently attached ligands:

a grid-based approach and an approach using exible sidechains.
The results are shown in Figure 3 for docking of penicillin in a
covalent complex with D-alanyl-D-alanine carboxypeptidase. In
the rst two experiments, we created a Gaussian map centered
on the CB or OG atoms of SER62 (Figs. 3A and 3B). We then
performed docking experiments with the appropriate atoms in
the ligand targeted to that map. Poor results were obtained: a
wide range of conformations was found and the conformation of
best energy showed only slight resemblance to the crystallographic conformation. Better results were obtained when two
Gaussian maps were used, corresponding to CB and OG of the
serine (Fig. 3C). This forced the docked ligand to adopt the
appropriate geometry of bonding with the serine, and resulted in
docked conformations that were much more similar to the crystallographic conformation.
Use of a exible sidechain to model the covalent ligand gave
excellent results. All 10 docking runs gave similar conformations, all of which were very similar to the crystallographic conformation (Fig. 3D).

Conclusions
Dependence on grid-based energy evaluation is a major limitation of AutoDock4. It is required to allow rapid evaluation of
binding energies during the docking simulation, but it places a

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severe restriction on the representation of the target macromolecule: all of the atoms included in the grid must be treated as
rigid. The off-grid modeling of specic sidechains is a method
for incorporating limited exibility within this paradigm, and the
results presented here show that it will be effective in some
cases. However, adding exibility presents several problems: (1)
the calculation of the receptor energy is more computationally
intensive since exible regions must be evaluated by a full pairwise energy evaluation, and (2) the conformational space is
larger, and hence, there is more potential for false positives.
Future solutions to these problems will require advances in energetic functions, simplifying and/or speeding up pair wise
approaches, and use of hierarchical approaches19,20 that allow
different levels of sophistication in a docking simulation.

Acknowledgments
This is manuscript 19885 from the Scripps Research Institute.

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