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J Comput Chem. 2009 December ; 30(16): 2785–2791. doi:10.1002/jcc.21256.

AutoDock4 and AutoDockTools4: Automated Docking with

Selective Receptor Flexibility
Garrett M. Morris1, Ruth Huey1, William Lindstrom1, Michel F. Sanner1, Richard K. Belew2,
David S. Goodsell1, and Arthur J. Olson1,3
1 Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037

2 Department of Cognitive Science, University of California at San Diego, La Jolla, CA 92093

Abstract
We describe the testing and release of AutoDock4 and the accompanying graphical user interface
AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are
reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a
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cross-docking experiment using flexible 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
modification of the flexible sidechain technique.

Keywords
AutoDock; computational docking; protein flexibility; covalent ligands; computer-aided drug
design

INTRODUCTION
Automated docking is widely used for 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 1–8.
AutoDock combines an empirical free energy force field with a Lamarckian Genetic
Algorithm, providing fast prediction of bound conformations with predicted free energies of
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association9.

In our hands, AutoDock3 has proven to be effective in roughly half of complexes that we
have studied. The remaining half show significant 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 here--
AutoDock4--incorporates explicit conformational modeling of specified sidechains in the
receptor to address this problem. This capability also provides an effective method for
analysis of covalently-attached ligands.

3
Corresponding author: Arthur J. Olson, Department of Molecular Biology – MB5, The Scripps Research Institute, 10550 N. Torrey
Pines Road, La Jolla, CA 92037, tel: 858 784 9702, fax: 858 784 2860, [email protected].
 Morris et al. Page 2

METHODS
Overview of AutoDock4
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Since its release in 199010, AutoDock has proven to be an effective tool capable of quickly
and accurately predicting bound conformations and binding energies of ligands with
macromolecular targets9,11–14. In order 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, finding local minima, and then
pass this information to later generations. A simulated annealing search method and a
traditional genetic algorithm search are also available in AutoDock4.
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AutoDock4 uses a semiempirical free energy force field to predict binding free energies of
small molecules to macromolecular targets. Development and testing of the force field has
been described elsewhere11. The force field 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 2–3
kcal/mol in prediction of binding free energy in cross-validation studies.

Receptor Flexibility
AutoDock4 allows fully flexible modeling of specific portions of the protein, in a similar
manner as the ligand. The user selects specific sidechains and these 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 flexible ligand. The remaining portion of the protein is represented using the
affinity grids described above.
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Methods for Covalent Docking

We have developed and tested two methods for docking of covalently-attached complexes: a
grid-based approach and a modification of the flexible 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 within the Gaussian well. One caveat is that this 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 define the bond that is
formed during covalent linkage. Note, however, that the conformational freedom allowed

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with a single Gaussian grid may be an advantage if the method is used, for instance, to target
ligands to metal coordination sites.
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We also tested use of the flexible sidechain method for docking of covalent ligands. In this
case, a coordinate file 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 flexible 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 fill this need.
AutoDockTools facilitates formatting input molecule files, with a set of methods that guide
the user through protonation, calculating charges, and specifying rotatable bonds in the
ligand and the protein (described below). 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
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experiments.

AutoDockTools is implemented in the object-oriented programming language Python and is

build from reusable software components15,16. 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 flexible 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 files 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 generic OpenGL-based 3-dimensional viewing
component; and MolKit, a hierarchical data representation of molecules. AutoDockTools
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consists of a set of commands dynamically extending PMV with commands specific to the
preparation, launching and analysis of AutoDock calculations. Hence, all PMV commands
(such as reading/writing files, 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 file that can be rerun. In summary, AutoDockTools is an example of a
specialization of the generic molecular viewer PMV for the specific application of
AutoDock.

Specification of Ligand and Receptor Flexibility in AutoDockTools

AutoDockTools provides an interactive method for defining the torsional tree for a given
ligand and receptor. Each step in this process has been automated to allow automatic

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assignment, for use in batch processes such as virtual screening. Ligand flexibility is
assigned in several steps. First, a root atom is chosen, which will act as the center of rotation
during coordinate transformation in the docking simulation. To find the optimal atom, we
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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 flexibility 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 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 previously11. 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 inconsistency 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 modified
manually, as described in the previous report.

The 87 HIV protease complexes were aligned to allow easy comparison of docked
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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 contacted a sidechain atom with less
than 2Å distance. Other 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 Å.

In the HIV cross dockings, ligand flexibility was limited to 10 torsional degrees of freedom,
picking 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
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complexes that included this water in the crystallographic structure, and hydrogen atoms
were added in geometry that allowed hydrogen bonding to the flaps.

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 solvated11. 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 artificial force field
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 artificially 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

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options in AutoDock allow the user to use an energy-minimized conformation of the ligand
as the unbound model.
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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://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.

RESULTS AND DISCUSSION

Redocking Analysis
Our first 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 calibration11. AutoDock4 successfully redocks most
complexes with about 10 or fewer torsional degrees of freedom, but fails for most
complexes with higher conformational flexibility. In 100 of 188 complexes, the docked
conformation with lowest energy was within 3.5Å RMSD of the crystallographic
conformation. This is slightly better than a similar redocking experiment with AutoDock3.
AutoDock3 successfully docked complexes with about 10 or fewer torsional degrees of
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freedom, and in 97 of 188 complexes, the lowest energy conformation showed RMSD less
than 3.5 Å (data not shown).

Cross Docking Analysis with Protein Flexibility

To validate the use of flexible 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.

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 flexible. 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
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conformations. The torsional degrees of freedom in the ligand were limited to 10, with an
added 6 degrees of freedom for the two arginine sidechains in the flexible sidechain tests.

The complexes in Figure 2 are arranged to highlight the structural features of the complexes.
The complexes 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 don’t 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 specificity in docking, although this is not observed.

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As seen in Figure 2A, flexible docking improves docking in the complexes that are most
expected to benefit. The white band below center corresponds to large inhibitors binding to
protease structures that were solved with small inhibitors. These complexes show the most
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favorable change in docking energy with flexible 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
through1hvj) are cases where large inhibitors are docked to active sites with ARG8 in a non-
permissive position, so these are the complexes where we expect improvement with a
flexible 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 find 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 degrees of freedom
are frozen in the proper orientation in the large inhibitors, and this tends to favor the
crystallographic conformation.
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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 find 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 flexibility 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 (Figure 2C).
Unfortunately, the experiments with flexible sidechains showed significantly worse results
for the small inhibitors, failing in over half of the cases. This underscores another limitation
of use of flexible 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.
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Covalent Docking
We tested two methods for docking covalently-attached ligands: a grid-based approach and
an approach using flexible sidechains. The results are shown in Figure 3 for docking of
penicillin in a covalent complex with D-alanyl-D-alanine carboxypeptidase. In the first two
experiments, we created a Gaussian map centered on the CB or OG atoms of SER62 (Figure
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 conformation 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 (Figure 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.

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Use of a flexible 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 (Figure 3D).
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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 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 specific sidechains is
a method for incorporating limited flexibility within this paradigm, and the results presented
here show that it will be effective in some cases. However, adding flexibility presents
several problems: 1) the calculation of the receptor energy is more computationally-
intensive since flexible regions must be evaluated by a full pair wise 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 hierarchal approaches19,20 that allow
different levels of sophistication in a docking simulation.

Acknowledgments
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This work was supported under grant RO1 GM069832 from the National Institutes of Health. We thank the
FightAIDS@Home volunteers and World Community Grid for computer resources. This is manuscript 19885 from
the Scripps Research Institute.

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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 3.5Å
RMSD relative to the experimentally observed conformation. Dots are complexes where the
best docked conformation was greater than 3.5Å RMSD from the experimental structure.
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Figure 2. Cross docking results

(A) Difference in energy between rigid docking and flexible docking. White points are
docking experiments where the flexible 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

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 Morris et al. Page 11

ordered similarly. (B) Cross docking with rigid protease structures. Each point is colored by
the RMSD of ligand atoms from the crystallographic structure, with RMSD=0 Å in white
and RMSD>5 Å in black. (C) Cross docking with ARG8 treated as flexible in the protease.
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Each point is colored with the same scale as in (B).

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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 flexible sidechain to
model the covalent ligand.
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