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https://doi.org/10.1038/s41596-021-00597-z

A practical guide to large-scale docking

Brian J. Bender1,4, Stefan Gahbauer 1,4, Andreas Luttens 2, Jiankun Lyu1, Chase M. Webb1,
Reed M. Stein1, Elissa A. Fink1, Trent E. Balius 3, Jens Carlsson2, John J. Irwin1 and
Brian K. Shoichet1 ✉
Structure-based docking screens of large compound libraries have become common in early drug and probe discovery. As
computer efficiency has improved and compound libraries have grown, the ability to screen hundreds of millions, and even
billions, of compounds has become feasible for modest-sized computer clusters. This allows the rapid and cost-effective
exploration and categorization of vast chemical space into a subset enriched with potential hits for a given target. To
accomplish this goal at speed, approximations are used that result in undersampling of possible configurations and
inaccurate predictions of absolute binding energies. Accordingly, it is important to establish controls, as are common in
other fields, to enhance the likelihood of success in spite of these challenges. Here we outline best practices and control
docking calculations that help evaluate docking parameters for a given target prior to undertaking a large-scale
prospective screen, with exemplification in one particular target, the melatonin receptor, where following this procedure
led to direct docking hits with activities in the subnanomolar range. Additional controls are suggested to ensure specific
activity for experimentally validated hit compounds. These guidelines should be useful regardless of the docking software
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used. Docking software described in the outlined protocol (DOCK3.7) is made freely available for academic research to
explore new hits for a range of targets.

Introduction
Screening chemical libraries using biophysical assays has long been the dominant approach to dis-
cover new chemotypes for chemical biology and drug discovery. High-throughput screening (HTS) of
libraries of 500,000 to 3 million molecules has been used since the 1990s1, and multiple drugs have
had their origins in this technique2. While the libraries physically screened in HTS were an enormous
expansion on those used by classical, pre-molecular pharmacology3, they nevertheless represent only
a tiny fraction of possible ‘drug-like’ molecules4. DNA-encoded libraries5, where molecules are
synthesized on DNA that encodes their chemistry, begin to address this problem by offering
investigators libraries of 108 molecules, sometimes more, in a single, highly compact format; and
multiple such libraries can be used in a single campaign. However, as DNA-encoded libraries are
restricted to reactions on DNA, reaction chemistries are limited to aqueous solutions, thereby limiting
the type of chemical reactions and subsequent chemical libraries available with this technology6.
Computational approaches using virtual libraries are an attractive way to explore a much larger
chemical space7. Large numbers of molecules—certainly into the tens of billions, and likely many
more—may be enumerated in a virtual library. Naturally, very few of these compounds can ever be
actually synthesized because of time, cost and storage limitations, but one can imagine a computa-
tional method to prioritize those that should be pursued. In practice, this idea has had two limitations
that have prevented wide-scale adoption: the virtual libraries have rarely been carefully curated for
true synthetic accessibility8, and there were well-founded concerns that computational methods, such
as molecular docking, were not accurate enough to prioritize true hits within this large space9.
In the last several years, however, two advances have at least partly addressed these problems:
First, several vendors and academic laboratories have introduced ‘make-on-demand’ libraries
based on relatively simple two- or three-component reactions where the final product is readily
purified in high yields10. At Enamine, a pioneer in this area, >140 reactions may be used to synthesize
products from among >120,000 distinct and often highly stereogenic building blocks, leading to a

1
Department of Pharmaceutical Chemistry, University of California–San Francisco, San Francisco, CA, USA. 2Science for Life Laboratory, Department of
Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. 3NCI RAS Initiative, Cancer Research Technology Program, Frederick National
Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, MD, USA. 4These authors contributed equally: Brian J. Bender,
Stefan Gahbauer. ✉e-mail: [email protected]

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remarkably diverse and, critically, pragmatically accessible library of currently over 29 billion
molecules11.
Second, structure-based molecular docking, for all of its problems, has proven able to prioritize
among these ultra-large libraries, if not at the tens of billion molecule level, then at the 0.1–1.4 billion
molecule level12–14, finding unusually potent and selective molecules against several unrelated targets
(Table 1). Indeed, simulations and proof-of-concept experiments suggest that, at least for now, as the
libraries get bigger, docking results and experimental molecular efficacies improve12.
If docking ultra-large libraries brings new opportunities, it also brings new challenges. Docking
tests the fit of each library molecule in a protein binding site in a process that often involves sampling
hundreds-of-thousands to millions of possible configurations. Each molecule is scored for fit using
one of several different scoring functions15–18. To be feasible for a billion-molecule library on
moderately sized computer clusters (e.g., 500–1,000 cores), this calculation must consume not much
more than 1 s/molecule/core (1 ms/configuration).
This need for speed means that the calculation cannot afford the level of detail and number of
interaction terms that would be necessary to achieve chemical accuracy. For instance, docking
typically undersamples conformational states, ignores important terms (e.g., ligand strain) and
approximates terms that it does include (e.g., fixed potentials)19,20.
Owing to these approximations and neglected terms, docking energies have known errors, and the
method cannot even reliably rank order molecules from a large library screen21,22. What it can hope
to do, however, is separate a tiny fraction of plausible ligands from the much larger number of library
molecules that are unlikely to bind a target. This level of prioritization is enhanced with the careful
implementation of best-practice guidelines and controls. It is the goal of this essay to provide
investigators with such best practices and control calculations for ultra-large library docking, though
they equally apply to modest-sized library docking (Table 1). These will not ensure the success of a
prospective docking campaign—the only true test of the method—but they may eliminate some of the
more common reasons for failure.
We begin by describing protocols and controls that can be used across docking programs and that
are general to the field (Fig. 1). There are by now multiple widely used and effective docking
programs23–31, employing different strategies for sampling ligand orientations and ligand con-
formations in the protein site, for handling protein flexibility, and for scoring ligand fit once they have
been docked. Notwithstanding these differences, there are strategies and controls that may be used
across docking programs, including how to prepare protein sites for docking calculations, bench-
marking controls to investigate whether one can identify known ligands from among a large library,
and controls to investigate whether one’s calculations contain biases towards particular types of
interactions. Since the true success of a virtual screen is the experimental confirmation of docking
hits, we also propose a set of control assays to validate initial in vitro results. We then turn to
protocols that are specific to the docking program we use in our own laboratory, DOCK3.7—these
necessarily get into fine details, and will be of most interest to investigators wanting to use this
particular program.

General guidelines for virtual structure-based drug discovery

Structure preparation and suitability for docking
Any structure-based campaign begins with a suitable target site. The most promising starting point
for a virtual screening campaign is typically a high-resolution ligand-bound structure. Ligand-bound
(holo) structures usually outperform ligand-free (apo) structures as the geometries of the binding
pocket are better defined in the bound state than in the unbound state32,33. If there is no available holo
structure, tools such as SphGen34, SiteMap35 and FTMap36 can be used to identify potential ligand
binding sites.
Generally, small, enclosed binding pockets that well complement a ligand perform better than large,
flat and solvent-exposed binding sites typical of protein–peptide or protein–protein interactions. For
instance, neurotransmitter G-protein-coupled receptor (GPCR) orthosteric sites, such as the β2
adrenergic, D4 dopamine, histamine H1 and A2a adenosine receptors37–40 typically have higher hit rates
and more potent docking hits than do peptide receptors like the CXCR4 receptor41, and these often
perform better than the more open sites typical of soluble enzymes like β-lactamase12,42. In most cases,
targeting protein–protein interaction surfaces, outside of a few that are well defined43, often leads to
disappointing results.

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Table 1 | Some recent large-scale docking campaigns

Screen target Library size/type Best hit Docking rank Docking program Computing time Hit rate (hits/
molecules
tested)

D4 dopamine 138 million ZINC621433144 Top 0.07% DOCK3.7 4.2 × 104 core h 24% (58/238)
receptors12,37 (make-on-demand) EC50 = 180 pM (1 s/molecule)

N
HN
N
O

600,000 Compound 9 Top 0.33% DOCK3.7 Not reported 20% (2/10)

(in-stock) EC50 = 213 nM
O
F OH
S
H
N
N
H

AmpC 99 million ZINC339204163 Top 0.00001% DOCK3.779 4.1 × 104 core h 11% (5/44)
β-lactamase12,119 (make-on-demand) Ki = 1.3 µM

O H
N OH
S
O O
OH
Cl
69,000 Compound 3 Not reported DOCK3.5120 Not reported 12.5% (2/16)
(HTS library) Ki = 37 µM

O O
OH
HO

OH
O O

MT1 Melatonin 150 million ZINC442850041 Top DOCK3.779 45,020 core h 39% (15/38)
receptor13 (make-on-demand) EC50 = 470 pM 0.005%
N

O
O N

14
KEAP1 1 billion iKeap1 Top 0.0001% QuickVina 2121 30 s/molecule 11.7% (69/
(make-on-demand) Kd = 114 nM (initial screen) 590)
Smina Vinardo122
O and AutoDock
N N O
S Vina23
O
(secondary
N N O screen)
O S O

A2 adenosine 4 million Compound 9 Within ICM docking 30 s/molecule 41% (23/56)

receptor38 (commercially Ki = 32 nM top 0.05% and Screening123
available)
NH2
O N
N O
N
HN

Cl Table continued

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Table 1 (continued)
Screen target Library size/type Best hit Docking rank Docking program Computing time Hit rate (hits/
molecules
tested)

HisG124 500,000 Compound 4 Within GOLD125 (initial ~60 h on 350 14% (7/50)
(commercially Ki = 2 µM top 0.2% screen) FLEXX24 networked PCs
available) (secondary
screen of
top 1%)
N
N
H
N S
N S
O N

Dipeptidyl 190,000 Compound 1 Within GLIDE25 (initial Not reported 15% (15/99)
peptidase IV (commercially IC50 = 5.77 µM top 0.05% screen)
(DPP4)126 available) AutoDock4 26

H (secondary
N screen of top 2%)
HO
N

N
N
O H

Docking rank indicates how highly ranked the best hit was among all virtually screened compounds. Hit rate denotes the fraction of docking hits confirmed in physical experiments.

Protein target Structure preparation Docking parameters

100
Control calculations
LogAUC ZINC20 Molecular weight (amu)
Default: 221 200 250 300 325
Optimized 1: 14.84
%ligands found 75 Optimized 2: 24.22
–1 40 K 97 K 1.3 M 1.9 M
Optimized 3: 36.40 0 192 K 937 K 6.8 M 10 M
50 1 516 K 3.8 M 21 M 33 M

2 723 K 8.8 M 32 M 48 M

cLogP
25
2.5 277 K 4.3 M 19 M 32 M

3 151 K 3.3 M 17 M 12 M
0
10–1 100 101 102 3.5 63 K 1.4 M 16 M 24 M
%decoys found
4 Virtual compound
Optimization and evaluation of
4.5 library
Concentration–response
virtual screening
curves
100 Compound 1 Docked compounds 109
Compound 2
80 Compound 3
Top 0.1% 105 Docking
60
Signal

program
Filter1
40

20 Filter2
Crystal structure
Cryo-EM structure 0 Hits 50–100
Homology model –9 –8 –7 –6 –5 –4 Large-scale
log(compound)
Hit-picking docking screen
Experimental tests

Fig. 1 | Large library docking workflow. The two required inputs for such a screen are the target structure and a screening database. Prior to using the
database, the target structure must be converted into a representation used by the docking software and the pocket should be optimized with control
calculations using retrospective analysis on known actives. After the prospective library has been docked, top-ranked hits can be filtered and selected
for experiment. Multiple assays and controls are typically necessary to confirm activity.

Modifying the high-resolution protein structure. It is not always a good idea to use the structure exactly
as it was found in the database.
● Dealing with mutations. For stability, crystallization and other biochemical reasons, high-resolution

protein structures are sometimes determined in a mutant form; such mutations should be reverted to
the wild type especially if they are within the ligand site to be targeted. Missing side chains and loops in
the experimental structures should be added as well if they are close to the binding site, while those that
are weakly defined by the experimental observables (e.g., low occupancy, high displacement parameters
(B values), poor electron density) should be examined critically
● What about water molecules? When the resolution permits, water molecules can also be included in

the target preparation, often treating them as nondisplaceable parts of the protein structure. Typically,
water molecules enclosed in the targeted binding pocket or involved in interactions between the

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co-crystallized ligand should be considered as they may determine side-chain conformations or offer
additional hydrogen-bonding sites. Some docking programs will allow water to be switched on or off
during the docking44 or include other implicit solvent terms45
● Buffer components. Buffer components such as PEG and salts are likely specific to the crystallization

conditions, and should be removed

● Cofactors. Cofactors like heme or metal ions should be considered if they are involved in ligand recognition

● Hydrogen atoms. Due to the resolution limits of most experimentally determined structures, hydrogen atoms

are often unresolved. The position of many hydrogen atoms can be readily modeled according to holonomic
constraints (e.g., backbone amide hydrogen atoms). For those that are not, such as hydroxyl protons on serine
and tyrosine residues, imidazole hydrogens on histidine, and the adjustment of frequently erroneous terminal
amide groups for glutamine and asparagine residues46, programs like Reduce47 (default in DOCK3.7), Maestro
(Schrödinger)48, PropKa49 or Chimera50 can be used to protonate the target of interest. While these automated
protocols usually produce reasonable protonation states, special care should be taken for the residues within
the ligand binding pocket or residues that form a catalytic/enzymatic site. Lastly, although less frequently
encountered, glutamic and aspartic acids can adopt protonated forms under specific circumstances51
● In summary, the protonation of the protein structure is critical for the success of docking to more accurately

depict the Van der Waals (VDW) surface and dipole moments of the binding pocket

Homology modeling
When no experimental structure has been determined for the target protein, structural models can be
generated if a template structure with high sequence identity is known. Common programs used for
homology modeling include Modeller52, Rosetta53, ICM27 and I-Tasser54. These two principles can
improve the chances of success:
● Typically, the higher the sequence identity between the target and the template, the better the accuracy

of the model55. Particular focus should be given to identity within the target binding pocket; if there is
a choice, choose the template that has the highest identity in the binding pocket
● Incorporation of a ligand during the modeling process or ligand-steered homology modeling

approaches56,57 will help prevent the pocket from collapsing inward, and will better orient the side
chains of binding residues32,58,59
When it is unknown how a ligand binds within the pocket, orthogonal experimental data can
guide the modeling such as iterating between docking and modeling60,61. In the case of MRGPRX262
and GPR6863, for instance, the authors predicted multiple binding poses of a known active ligand and
used mutagenesis and binding assays to test these predictions. A binding mode and receptor structure
were identified that was consistent with the mutagenesis data and used for subsequent preparation in
the prospective screen. Despite many difficulties, homology models have been successful in identi-
fying novel ligands from prospective docking campaigns41,62–76; though it is also true that, given the
choice, most investigators will prefer to use a well-determined experimental structure.

Control calculations
Docking undersamples ligand–protein configurations and conformations, and its scoring of these
configurations for fit remains highly approximate. Unlike methods like quantum mechanics, or
certain lattice calculations in statistical mechanics, docking has surrendered ‘ground truth’ to be able
to pragmatically search among a large and growing chemical space. Accordingly, control calculations
are critical to the success of a docking campaign. As with experimental controls, they do not ensure
prospective success, but they do guard against obvious sources of failure, and can help one under-
stand where things have gone wrong if they do. Through a key control, we assess whether the
prepared binding pocket and docking parameters can prioritize known ligands over presumed
inactive molecules. In an optimized binding pocket, these known actives should rank higher against a
background of decoy molecules in a retrospective screen, and reasonable poses should be predicted.
As it is more likely to know true actives than true inactives, it is common practice to use property-
matched decoys77, which are compounds that have similar physical properties as the actives, but
unrelated topologies, and so are presumed inactive. The DUDE-Z server (tldr.docking.org) was built
specifically to generate decoys for a given list of ligands by matching the following physical properties:
molecular weight, hydrophobicity (LogP), charge, the number of rotatable bonds, and the number of
hydrogen bond donors and acceptors78.
The performance of the parameterized binding pocket to enrich known ligands over decoys can be
evaluated by receiver-operator characteristic (ROC) curves, quantifying the true positive rate as a
function of false positive rate (Fig. 2)17,77,79–81. The area under the curve (AUC) is a well-regarded

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a ROC curve b Semilogarithmic ROC curve

100 100
Model: AUC Model: LogAUC
Model 1: 73.21 Model 1: 14.84
Model 2: 75.98 Model 2: 36.40

75 75
%ligands found

%ligands found
50 50

Early enrichment
25 25

0 0
0 25 50 75 100 10–1 100 101 102
%decoys found
%decoys found (log)

Fig. 2 | Enrichment of actives against decoys. a, ROC curves for two models used for retrospective docking screens plotting the rate of true positives
found against decoys found. The AUC can be used to describe the ability of the models to identify true positive, known ligands against a background of
decoys. In this format, the two models have similar AUCs, suggesting similar performance. b, Semilogarithmic ROC curves focus on the early
enrichment, i.e., determine if true positives are identified within the e.g. top 10% (gray area) of docked decoys. The LogAUC is calculated as the
difference between the semilogarithmic AUC of the model and the random semilogarithmic AUC (dashed line). In this format, it is clear that model 2
outperforms model 1 in early enrichment with a LogAUC value more than double of model 1.

Decoy set MWT LogP Charge # HBA/HBD Topology # of decoys Goal

Property-matched decoys Matched to Matched to Matched to Matched to

Unmatched 50/active Measure enrichment
(DUDE-Z) actives actives actives actives
Matched to Matched to Matched to Simulate performance in
In-stock set Any Any ~106
actives actives actives prospective screen
Matched to Matched to
Extrema set –2,–1,0,+1,+2 Any Any 103–106 Test for bias
actives actives
Measure real-world
Physical screen inactives Any ~103
performance

Fig. 3 | Control sets for retrospective docking calculations. For DUDE-Z decoys, properties of the decoys are either forced to match (green) or be
different (red). Properties that are neither selected for or against are highlighted in yellow. In the Extrema set, the charge state is explicitly sampled.

metric to monitor the performance of a virtual screen by a single number17 (Fig. 2a). The log
transformation of the false positive rate enhances the effect of early enrichment for true positives17
(Fig. 2b). This is important because, in a docking campaign with hundreds of millions of molecules,
only compounds ranked within the top 0.1% are often closely evaluated (see ranks in Table 1).
For example, if a retrospective docking challenge shows that known actives are only identified starting
around the tenth percentile, novel actives may be missed in a prospective screen. In this setting,
higher LogAUC values correspond to better discrimination between actives and inactives and provide
a sanity check on the ability of the docking parameters to identify actives.
A second criterion is the pose fidelity of the docked ligands to their experimental structures.
The validity of predicted binding poses can be assessed qualitatively by visual inspection of
reported key interactions between protein and ligand or, in the best case, quantitatively by calculating
root mean square deviations between predicted and experimentally determined poses82. During pocket
modeling and docking parameter optimization, one will often insist that the retrospective controls lead
to both high LogAUC values and good pose fidelity; often there will be some trade-off between the two.
While calibrating the scoring functions, it is also important to monitor the contributions of each energy
term to the total score and ensure they match the properties of the binding pocket. If one term
dominates, the scoring may have been overoptimized to that term, while other protein–ligand inter-
actions are underweighted, leading to dominance by a certain type of molecule. For instance, if the
docking score of a polar solvent-exposed pocket is inappropriately dominated by VDW energies, large
molecules may score high due to nonspecific surface contacts with the target protein.
In addition to property-matched decoys, other chemical matter can be used to evaluate different
aspects of the docking model (Fig. 3). A test set including molecules with extreme physical properties

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(Extrema set), such as a wide variety of different net charges, can be screened to measure whether the
docking model succeeds in prioritizing molecules with net charges corresponding to known actives78. If
there is a difference in the charge of top-ranked compounds from the extrema set and the charges of the
known ligands, the scoring may be biased. In another useful control experiment, a small fraction of the
purchasable chemical space (e.g., readily available ‘in-stock’ compounds from multiple vendors)
representing the characteristics of the ultra-large make-on-demand screening library can be docked
against the protein model. This control serves two purposes: to test whether the positive control
molecules remain among the highest scored compounds and to examine if intriguing novel compounds
rise to the top of the rank-ordered docking list. It may even be fruitful to purchase and experimentally
test a few promising and structurally diverse in-stock compounds as it could inform the docker if the
binding pocket model is likely to find hits in the ultra-large library screen. If top ranked compounds do
not form expected interactions, further optimization may be beneficial.
Another control, if available, are true inactives from a previous discovery campaign. These can be
used as a background against known actives to provide a ‘real-world’ benchmark of the performance
of the system. Lastly, for a protein that has little or no known chemical matter (i.e., reported ligands),
enrichment calculations with known actives against a background of property-matched decoys may
be impossible. Here, the docking parameters can be calibrated by docking ‘Extrema’, which challenges
the docking with extremes of physical properties, and ‘in-stock’ compound sets, which probe how the
docking will perform on a representative subset of the library78,83. It remains true that, without
known ligands as positive controls, one is at a substantial disadvantage at setting up docking
campaigns, increasing the risk of failure.

Prospective screen
Once the docking model is calibrated, large libraries of molecules can be virtually screened against the
target protein. For this virtual screen, it makes sense to focus on compounds that are readily available for
testing. The ZINC20 database (http://zinc20.docking.org/) enumerates over 14 billion commercially
available chemical products, of which ~700 million are available with calculated 3D conformer libraries
ready for docking. Most of the enumerated compounds belong to the make-on-demand libraries of
Enamine and WuXi. Further, ZINC20 allows one to preselect subsets of molecules for docking, reducing
computation time. For instance, ZINC20 allows users to download ready-to-dock subsets of molecules
within user-defined ranges of molecular weight (MWT), LogP and net charge, as well as predefined
sets such as fragments (MWT ≤ 250 amu) or lead-like molecules (250 ≤ MWT < 350 amu; LogP ≤ 3.5).
The result of a prospective screen is a list of molecules rank-ordered by docking score.

Hit-picking
A well-controlled docking calculation can concentrate likely ligands among the top-ranked molecules.
But even if it was able to do so among the top 0.1% of the ranked library, in a screen of 1 billion
molecules this would still leave 1 million molecules to consider, and with the errors inherent in
docking, many of these will be false positives. Accordingly, we rarely pick the top N-ranked com-
pounds by docking to test experimentally, but rather will use additional filters to identify promising
hits within the top scoring 300,000–1,000,000 molecules. These filters can catch problematic features
missed by the primary docking function, ensure dissimilarity to known ligands and promote diversity
among the prioritized compounds.
Compounds may be filtered for both positive and negative features84 (Table 2). For instance, one
may insist that a docked orientation has favored interactions with key residues. Conversely, molecules
with strained conformations should be discarded. Molecules with unsatisfied, buried hydrogen-bond
donors and acceptors may also be deprioritized. Compounds with metabolic liabilities85,86 or that
closely resemble colloidal aggregators87 can also be filtered out, despite otherwise favorable scores.
Further, as closely related compounds will likely dock in similar poses with similar score, we typically
cluster compounds by 2D structure similarity after all other filters have been used and only select the
best scoring cluster representative for testing. In such a large dataset, clustering can be computa-
tionally expensive, so reduced scaffold clustering such as Bemis–Murcko88 analysis is useful to
efficiently parse the compounds. In principle, many of these filters could be included directly in the
scoring functions, but balancing them against other scoring terms can demand extensive optimization
and will likely increase the compute time, which will become a hindrance for ultra-large library
docking. Lastly, visual examination of the docked poses has been useful for selection of compounds to
purchase. Following the criteria in Table 2, we typically inspect up to 5,000 compounds visually after
applying automated filtering and clustering steps.

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Table 2 | Hit picking criteria

Docking score Docking scores are approximations of interaction energies and therefore cannot be used for absolute
ranking of hits. Its main purpose is to identify compounds that are likely to bind from the large
background of compounds that are unlikely to bind
Broken molecules Errors in 3D building can result in incorrectly built compounds. Common errors include improper
tautomerization or protonation states owing to the difficulty in predicting pKa. Such ‘broken’ molecules
should be deprioritized during visual inspection
Internal strain Often internal strain is not included in energy functions but is an important concern since a ligand may
score highly if it adopts a strained conformation that increases its contacts with the receptor (e.g.,
nonplanar amide). Visual examination can catch these strained compounds, but various tools have been
developed to systematically analyze hundreds or thousands of compounds making them useful in this
setting127–129. We recently proposed a computational tool that estimates torsion strain energies based on
experimentally determined torsional populations in the Cambridge Structural Dataset129,130
Interaction patterns Specific interactions to key residues are main features of promising candidates. Hydrogen bonds between
ligands and side chains can be computationally identified by simple distance cut-offs (<3.5 Å). For
example, hydrogen bonding to kinase hinge residues131, or salt bridges to the conserved aspartic acid
D3.32 in aminergic GPCRs132, offer reasonable anchor points for interaction filters. In a similar fashion, a
pharmacophore filter can be used to search for any atoms in an unexplored subpocket within the larger
binding pocket. Molecules that score well but form only one key interaction may be deprioritized.
‘Floating’ molecules that may bury an energetically frustrated water should also be eliminated
Unsatisfied hydrogen bond donors Molecules with unsatisfied hydrogen bond donors or acceptors, especially in hydrophobic pockets of the
and acceptors site, often pay a high desolvation cost; not all scoring functions are designed to filter these out explicitly.
Therefore, we suggest keeping the number of unsatisfied acceptors below 3 and the number of
unsatisfied donors to 1 or below, as burying a donor can incur a greater penalty than burying
an acceptor133
Novelty filter One of the advantages of large-scale docking with make-on-demand chemical libraries is the ability to
screen for novel chemistry. Therefore, if the goal of the project is to identify novel scaffolds for hits, it is
useful to filter out docking hits that resemble known actives. This can be accomplished using any number
of computational methods that compare the 2D or 3D topologies of hit compounds. In our hands, we
typically remove hits that have an ECFP4 Tanimoto cutoff of 0.35 or higher to any known active12,13
Scaffold clustering From the initial docking screen, a set of diverse scaffolds should be selected for experimental testing to
cover a larger subset of chemical space. Clustering metrics to identify diverse hits may include ECFP4134
fingerprints, Bemis-Murcko88 scaffolds, bcl::Cluster similarity135 or Atom Counts136

Experiments to test docking hits

The success of a docking campaign is ultimately measured by its ability to reveal novel chemotypes
that can be shown to experimentally bind to the target, typically in binding or functional assays
(Fig. 4). However, common artifacts should be controlled for: chemotypes likely to interfere with
specific assays89 (e.g., the controversial90 pan assay interference chemotypes), covalent adducts, redox
cyclers91 and aggregators87,92.
Among the most common of these mechanisms is colloidal aggregation, which can account for
>90% of all primary hits89,93. This aggregates sequester proteins with little selectivity, partially
denaturing and inhibiting them32,92,94–96, occasionally even activating them97,98, a common problem
both in HTS and also in docking screens92. Aggregators tend to have high LogP values and limited
aqueous solubility, so we prefer to dock and test molecules with LogP ≤ 3.5. Chemical stability or
reactivity can also contribute to experimental artifacts, and reactive scaffolds should be avoided.
The ZINC20 database allows users to select screening libraries in the lead-like chemical space (i.e.,
low LogP) and exclude reactive compounds9,99. Unless controlled for in the experimental setting
(Box 1), these artifacts can lead to false positives.
Once convinced that one’s hits are not artifactual, more detailed testing on-target is warranted. For
all targets, this involves determining concentration response curves, typically with 7–12 points at half-
log intervals, with the transition being sampled over at least two log orders of concentration (Fig. 4).
For enzymes, determining a true Ki will illuminate mechanism, though this can be laborious and it
might only make sense to do this for characteristic lead molecules. For receptors such as GPCRs and
ion channels, functional assays are typically performed to determine if the new ligand is acting as an
agonist, an antagonist or an allosteric modulator. For GPCRs, initial screening assays may differ from
secondary confirmatory assays, and showing that a molecule is active in more than one assay is often
quite useful.
Before initial hits are advanced for optimization, it is important for make-on-demand libraries to
confirm the identity of the hit compound. Limitations of virtual library enumeration, chemical

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Single concentration binding or functional assay

100
80

Signal
60
Primary screen

40
20
0
Compounds

Potential hits

Test for colloidal aggregation Confirmation of chemical identity

Dynamic light scattering (DLS) Concentration–response curve Mass spectrometry (MS)
Off-target enzyme inhibition (+/– detergent) (7–12 points, half-log intervals) Nuclear resonance spectroscopy (NMR)

Concentration-response
Off-target inhibition
curves
100 No detergent 100 Compound 1 22.0
With detergent Compound 2 20.0 NMR
NMR
80 80 Compound 3 18.0
Activity (%)

16.0
Hit validation

Signal

14.0
60 60 12.0
10.0
40 40 8.0
6.0
20 20 4.0
2.0
0.0
0 0
10.0 8.0 6.0 4.0 2.0 0.0
–9 –8 –7 –6 –5 –4 –9 –8 –7 –6 –5 –4 δ/ppm

log(compound) log(compound)

Confirmed hits

Analog-by-catalog
Affinity of purified stereoisomers Experimental high-resolution structure
focused SAR libraries
binding pose confirmation
Hit optimization and

Docking
Crystal structure

Fig. 4 | Suggested experimental validation of docking hits. In general, a primary screen will use a limited number of compound concentrations to test
for activity at a target. Compounds that pass a set threshold of activity in the primary screen will be moved forward to secondary confirmation of
activity that is not attributed to colloidal aggregation. Identity of the compound should be confirmed if it passes these stages and before proceeding to
optimization by stereoisomer purification, selection of analogs and/or experimental structure determination.

synthesis and downstream purification can result in mismatches between the in silico predicted
compound and the in vitro tested compound. For example, in the screen against the severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain protein, a number of purine
derivatives with an N9 substitution were requested but N3-substituted compounds were synthesized
instead83. Accordingly, it is worth fully characterizing promising compounds before costly lead
optimization is undertaken; we ourselves have made the mistake of designing analogs based on an in
silico starting scaffold that was different than the true hit delivered from the chemists. At a minimum,
the identity and purity of the compounds should be determined. High-resolution mass spectrometry
or quantitative 1D proton nuclear magnetic resonance spectroscopy can be used to detect gross
variation between the tested sample and the expected molecule; these data can be obtained from most
vendors, or the experiments could be performed independently.
The most direct experiment to test a docking pose prediction is the determination of an experi-
mental protein structure in complex with the docking hit (Fig. 4). Such structures illuminate crucial

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Box 1 | Control experiments to identify false positives among tested compounds

Mechanism Structural alert Control experiments

Covalent Presence of an electrophile Incubation of ligand with the target typically increases activity. For soluble
Useful if sought, but if unintended (e.g., Michael acceptor, α- proteins, pre-incubate ligand and target at a concentration higher than the
are an artifact halo-ketone, activated nitrile) EC50, and then dilute to below the EC50. If irreversible, activity will reflect
the pre-incubation concentration. If reversible, ligand activity will diminish
over time
Redox cycling Almost always Several chemotypes (e.g., Test in the presence and absence of a reductant or radical trap, like
an artifact rhodanines) β-mercaptoenthanol or ascorbic acid
Spectroscopic interference Assay Multiple chemotypes, often Spectroscopic interference will change linearly with compound
disruption via light absorption or highly conjugated concentration, following Beer’s law, rather than sigmoidally as in a binding
fluorescence137–140, or inhibition isotherm. Molecules that might be inhibiting a reporter enzyme used in the
of a reporter enzyme, like assay (e.g., luciferase) may be tested for inhibition against it
luciferase141
Colloidal aggregation Likely the Multiple chemotypes. It is Run the assay in the presence and absence of a non-ionic detergent like
most common single mechanism difficult to predict which Triton X-100 (for soluble proteins) or Tween-80 (for cellular assays). Right
of artifact in early discovery compounds will aggregate. shifts of two- to threefold or greater in EC50 on detergent addition suggest
LogP > 3.5 and similarity to compound aggregation. Detergent solutions should be made fresh daily.
known aggregators can Control for the effect of the detergent on a well-behaved, nonaggregating
capture some of these, ligand, which should be unaffected93,142,143
but many will be missed Test for the formation of particles >30 nm in radii by dynamic light
(http://advisor.bkslab.org/) scattering92,144
For assays that will not tolerate detergent, an inert protein can be added to
prophylactically coat the colloids, protecting the assayed target from
adsorption. BSA is commonly used, though most inert soluble proteins will
work. The inert protein should be present in mg/mL concentrations92,95
Colloids are liquid droplets of pure compound, and can typically be pelleted
in a microfuge after a 20 min spin at low temperature. Draw off the
supernatant and test for activity—if it is much reduced, the compound is
likely an aggregator32,96
As with covalent inhibitors, pre-incubation typically increases the activity of
a colloidal aggregator. Unlike covalent inhibitors, incubation at higher
enzyme concentrations will typically slow the onset of aggregate-based
inhibition, eventually eliminating it almost entirely92
Counter-screen against unrelated off-target model enzymes, with and
without detergent. Common enzymes include malate dehydrogenase
(commercially available) and AmpC β-lactamase (freely available from
the authors)145

details of ligand recognition, including adjustments of binding site residues or the docking hit in the
binding pocket12,100,101. The key question to answer with high-resolution structures is whether
docking worked for the right reasons, i.e., does the predicted binding mode agree with an experi-
mental structure? Previous studies suggest that hits from virtual screening generally compare fairly
well with experimental high-resolution structures; i.e., key anchor points are predicted cor-
rectly12,83,102,103. However, as docking is typically performed against rigid protein structures, con-
formational changes, especially in flexible loops, will complicate pose prediction104. Water-mediated
protein–ligand interactions are generally difficult to explore de novo45,105, though experimentally
determined and structurally conserved water molecules can be included in virtual screens100. Lastly,
there are few cases where docking hits showing on-target activity revealed binding positions at
unexpected and nontargeted subsites. For example, the β2 adrenergic receptor allosteric modulator
AS408 was predicted to bind to an extracellular subpocket but crystallographically determined to bind
to the membrane-facing surface of the receptor106. In contrast, the pose of an inverse agonist also
identified at the β2 adrenergic receptor from in silico docking39 was confirmed by X-ray crystal-
lography107, demonstrating the variability in outcomes even at the same target.
If the protein target can be readily expressed and purified in milligram quantities, crystallography
and cryo electron microscopy can guide hit discovery early in the campaign. In campaigns against
targets where high-resolution structures are more challenging to obtain (e.g., for transmembrane
receptors), an experimental protein–ligand complex might only be achievable for the most potent hit
compound. Nonetheless, it is usually worth the effort to confirm the predicted binding site and ligand
pose, or to identify unexpected interactions between the discovered hit and the target106.

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Next steps: selecting analogs for hit to lead optimization
In the fortunate event that the virtual screen was successful and docking hits were confirmed by
different experiments, the newly obtained scaffolds are blueprints for exploring structure–activity
relationships and lead optimization.
A concern about molecules from make-on-demand libraries is that they will initially be delivered
as racemic or stereomeric mixtures since the production of pure stereoisomers requires more
sophisticated synthesis routes. If confirmed hits were observed from stereomeric mixtures, the
measured potency of the mixture may be artifactually lower if only one stereoisomer is active at the
target (e.g., the concentration of the active stereoisomer is less than the total mixture concentration).
Purified stereoisomers can typically be purchased from the make-on-demand compound supplier or
separated in-house.
Hit optimization can be performed in several ways. Synthetic chemistry groups may obtain
synthesis routes of the parent compound from the supplier allowing the generation of medicinal
chemistry-inspired analog series108. Alternatively, chemoinformatic tools can be employed to virtually
search the purchasable chemical space for structurally related scaffolds or identifying molecules with
a common substructure to the parent hit compound (analog-by-catalog). Searching for similar
molecules within the Enamine REAL space can be conducted using the SmallWorld (sw.docking.org)
search engine99. Molecules with an identical substructure as the parent compound can be found using
Arthor (arthor.docking.org)99. Additionally, the supplier of the parent compound might be able to
provide a collection of molecules resembling the hit scaffold. While we cannot provide clear guidance
or a best-practice protocol for hit-to-lead optimization, analog-by-catalog of docking hits obtained for
the melatonin 1 (MT1) receptor and AmpC led to compounds with improved potencies compared
with the initial parent compounds12,13. Currently, we recommend subtle changes to the starting
compound or modifications to test particular interactions as suggested from the docked pose.
It is our hope that the above guidelines will be useful for outlining a docking campaign from start
to finish. As shown, the prospective docking step is actually only one small component of the overall
pipeline (Fig. 1). Control calculations using retrospective datasets and docking setup optimization
make up the bulk of the process. Hit picking from the prospective screen requires careful perusal of
the data, and experimental design of the confirmatory assays is critical for defining success of the
campaign. We ourselves find that these guidelines insulate against the more common sources of
failure in large library docking campaigns. Lastly, we want to mention that docking campaigns against
protein targets without experimental structure, i.e., requiring homology modelling, or without known
active chemical matter for retrospective control calculations are particularly risky and should not
be initiated naively.
This concludes our general guidelines for large library docking. In the last section, we turn to a
detailed protocol to set up, optimize and prospectively screen a target of interest using DOCK3.7,
though any docking program can use the controls presented.
While the protocol should be general for most proteins, we provide example data from a recent
campaign against the MT1 receptor13, a target particularly well suited for docking: a crystal structure
had been determined; the orthosteric pocket is compact and almost completely encloses the ligands,
simplifying the biophysics; many such ligands exist for retrospective calculations and for optimiza-
tion; and in vitro assays to test docking hits were well established. We note that the MT1 receptor is
ideal for large library docking, and so the achieved hit rate, and the hit potencies, were unusually high.
Still, the docking optimization strategies below have been useful against a wide spectrum of targets,
including the Nsp3 macrodomain of SARS-CoV-283 and highly solvated pockets as in β-lactamase12,
and were developed against the spectrum of targets in the DUD-E benchmark77, ranging from ion
channels to kinases to soluble enzymes. It should be clear that the optimization strategies sketched
remain rooted in retrospective controls, and so will work best against targets with precedented
ligands, and against targets with well-formed and readily liganded binding sites.

Docking campaigns with DOCK3.7 and ZINC20

In DOCK3.7, ligands are placed in the target pocket by mapping ligand atoms onto predefined hot-
spots, so-called matching spheres. Matching spheres are generated from the coordinates of heavy
atoms from an input bound ligand structure, if available, and supplemented with coordinates based
on the negative image of the binding site generated from the program SphGen34. Ligand rigid
fragment (e.g., rings) are mapped onto matching spheres using a bipartite graph algorithm79,109.
Different ligand conformations and orientations are sampled in the binding pocket using

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precalculated 3D conformer libraries (flexibases, db2 files)9,99. During conformer library building,
each molecule is divided into its rigid fragments, and different conformations of rigid fragment
substituents are generated.
Ligand poses are evaluated using a physics-based scoring function (Escore) combining VDW,
electrostatic (ES) and ligand desolvation (lig_desol) energy terms (Equation 1):

Escore ¼ EVDW þ EES þ Elig desol ð1Þ

In order to allow rapid scoring of new poses (typically 1,000 poses per molecule per second),
contributions of the target protein pocket are mapped onto pregenerated scoring grids. The VDW
surface of the binding pocket is converted into a scoring grid by ChemGrid15. Electrostatic potentials
within the binding pocket are estimated by numerically solving the Poisson–Boltzmann equation
using QNIFFT16,110. Context-dependent desolvation energy scoring grids, both polar and apolar, are
generated by the Solvmap program17. Thereby, ligand desolvation energies are computed as the sum
of the atomic desolvation of each ligand atom scaled by the extent to which it is buried within the
binding site. Atomic desolvation energies for ligand atoms are calculated during ligand building using
AMSOL111 and are included in the ligand conformer library. Both, the electrostatic and ligand
desolvation scoring grids depend on the dielectric boundary between the low dielectric protein
(εr = 2) and high dielectric solvent (εr = 80) environments. Consequently, these scoring grids can be
fine-tuned by modulating the protein–solvent dielectric interface (see below Steps 41–44).
The following protocol (Fig. 5) prepares a starting structure (Steps 1–4), generates the scoring
grids and matching spheres (Steps 5–10), and collects a set of control ligands for model evaluation
(Steps 11–33). Control ligands are docked retrospectively (Steps 34–58) to determine the model’s
ability to identify actives from a pool of inactives. Optimization of sampling (Steps 59–64) and
scoring (Steps 65–77) is evaluated with the same controls. Model biases are evaluated using an
Extrema control set (Steps 78–88) to examine for charge preferences in the scoring grids and with a
small-scale in-stock screen (Steps 89–97) to ensure a computationally expensive large-scale pro-
spective screen (Steps 98–103) is likely to yield interesting chemical matter. Hit picking (Steps
104–108), though critical to success of these prospective experiments, is beyond the scope of the
protocol as it is highly user- and target-specific, and we suggest referring to Table 2 for insight.
Additionally, we caution against regarding hits from a screen as true positives until common artifacts
are ruled out (Box 1). All controls in silico and in vitro are applicable to any docking program or
computer-aided drug discovery campaign. The details of grid preparation and modification are
DOCK3.7-specific, but the principles are transferrable to other software.

Materials

Software
● DOCK3.7.5: apply for a license from http://dock.docking.org/Online_Licensing/dock_license_applica
tion.html. Licenses are free for nonprofit academic research. Once your application is approved, you
will be directed to a download for the source code. The code should run without issue on most Linux
environments, but can be optimized by recompiling with gfortran if needed. Questions related to
installation can be addressed to [email protected]
● Python: the current code uses python2.7, which will also need to be installed. Additional python

dependencies that are required for running scripts can be found in the file $DOCKBASE/install/
environ/python/requirements.txt
● AMSOL: free academic licenses can be obtained from https://comp.chem.umn.edu/amsol/

● (Optional) 3D ligand building software: if interested in 3D ligand building in-house (not necessary for

this protocol), licenses will also need to be obtained for ChemAxon (https://chemaxon.com/), OpenEye
Omega (https://www.eyesopen.com/omega) and Corina (https://www.mn-am.com/products/corina).
We note that, for many campaigns, 3D molecular structures with all necessary physical properties may
be downloaded directly from ZINC20. This tutorial makes use of a webserver for 3D ligand building
that is suitable for small control sets. Please apply for an account at https://tldr.docking.org/, which is
free of charge
● Chimera: this application is recommended for grid visualization with the VolumeViewer feature (see

Troubleshooting). The ViewDock feature, also within Chimera, is useful to examine the docking results

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Receptor structure preparation Ligand control set

X-ray, cryo-EM, model? Literature reports

Holo state or apo state? ChEMBL
Revert mutations? ZINC
Steps 1–10 Build missing residues? Patents Steps 11–33
Cap termini? In-house screening hits
Protonate manually?

Calculate input parameters Decoy sets

Electrostatics grid Property-matched decoys

Van der waals grid Extrema set
Ligand desolvation grid In-stock set
Matching spheres Known inactives

Steps 34–58 Retrospective control No known actives?

calculations

Binding poses of actives

LogAUC
Property-matched decoys Charge distribution
Energy distribution

Steps 59–64 Optimize sampling

Property-matched decoys Matching sphere scan

Steps 65–68 Optimize scoring

Property-matched decoys Boundary sphere scan

Steps 69–77 Polarize residues

Steps 78–88 Measure bias

Extrema set

Steps 89–97 Test prospective

performance

In-stock set
Test post-docking filters
Known inactives

Steps 98–103 Large-scale docking

Zinc database

Steps 104–108
Table 2 Hit picking

Post-docking filters
Visual hit picking

Experimental testing

Box 1 Hit validation

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Fig. 5 | Outline of the procedure for DOCK3.7 virtual ligand discovery campaigns. Collecting and preparing materials (blue) requires obtaining a
structure or model and ligand control sets and setting them up for retrospective control calculations (yellow). In each control calculation, modifications
may demand returning to a previous step and reoptimizing. In the absence of known actives for robust retrospective analysis, one may jump to testing
the prospective performance with a small library. With a final setup, large-scale prospective screening (orange) can proceed, followed by in vitro
testing of docking hits (green). The numbers refer to steps described in the Procedure.

Equipment
● Hardware: initial tests of this tutorial (grid preparation and small retrospective screens) can be
performed on a single workstation, but more intensive docking will need access to a high-performance
computing cluster (we regularly use 1,000 cores)
● Queuing system: DOCK3.7 comes with submission scripts for SGE, Slurm and PBS job schedulers.

If using a different scheduler, scripts may need to be adapted for your specific computing cluster
● Protein structure: structures can be downloaded from www.rcsb.org or generated by the user in the

case of homology models

● Example data: an example set of files used in this protocol including ligand and decoy sets, default

docking grids and optimized docking grids can be downloaded from http://files.docking.org/dock/
mt1_protocol.tar.gz. The example dataset uses the MT1 structure (PDB: 6ME3) co-crystallized with
2-phenylmelatonin

Equipment setup
Define the pathway to critical software
The environment will need to be defined in order to run the following commands correctly. The two
most important paths that need to be defined are to DOCKBASE and python. We provide an envir-
onment script in the example dataset that will need to be modified for a user’s settings. Always run the
following command with updated paths specific to your file directory before working with DOCK3.7:

source env.csh

Procedure
Section 1: set up binding pocket for docking calculations ● Timing 30 min to 1 h
1 Download the structure from the PDB or use in-house structures. Preference should be given to
structures of high resolution and with a ligand bound in the site that will be docked into. Apo
structures are useable, but tend to yield poorer results due to unconstrained binding site geometries32.
2 Visualize the binding pocket in PyMOL or Chimera to decide on the relevance of protein cofactors.
● Delete components from the structure that do not contribute to ligand binding such as lipids,

water molecules and buffer components

● Fusion proteins engineered for protein stability that are near the binding pocket should be

removed, and the resulting chain break should be either capped or the loop remodeled
● Protein cofactors such as heme or ions should also be kept or discarded depending on their

relevance to ligand binding in the pocket

● Additionally, examine the residues in the binding pocket for incomplete sidechain, multiple

rotamers or engineered mutations, and revert or rebuild as necessary

● It is helpful to examine the pocket with the electron density if available for making these

decisions. For the example MT1, we deleted the fusion protein, capped non-native termini and
reverted two mutations in the binding pocket (6me3_cleaned.pdb)
3 Save the rec.pdb file. This file will contain any component that will remain static during the
docking such as structural waters and cofactors.
4 Save the xtal-lig.pdb file. This file will contain the atoms of the bound ligand, which will be
used to generate the matching spheres that guide ligand sampling in the pocket.
5 Generate the scoring grids and matching spheres with blastermaster from these two inputs
(rec.pdb and xtal-lig.pdb)

$DOCKBASE/proteins/blastermaster/blastermaster.py --addhOptions="
-HIS -FLIPs " –v

? TROUBLESHOOTING

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6 Inspect the output, in particular the rec.crg.pdb file located in the new working directory to
ensure protonation of polar residues and side chain flips of glutamine and asparagine side chains
look accurate. Examine histidine tautomers (HID, HIE and HIP) as well. If everything looks as it
should, proceed to Step 10; otherwise, proceed to Step 7.

Generating a rec.crg.pdb file manually

7 If the automatically protonated rec.crg.pdb file does not fit to the expected protonation state of
key residues, the rec.crg.pdb file can be generated manually using various protein modeling
software packages including Rosetta112, Chimera50 or Maestro113. This may include manually
flipping side chain rotamers and setting the pH value for pKa calculation of charged residues. After
the modeling step is completed, new rec.pdb, rec.crg.pdb and xtal-lig.pdb files need
to be provided to blastermaster. To be compatible with the united atom AMBER force field, rec.
crg.pdb should only contain polar hydrogen atoms, and explicit histidine tautomer names. rec.
pdb and xtal-lig.pdb only need to contain heavy atom coordinates. In Step 8, we provide a
script that produces the required files.
8 Assuming the protein modeling software resulted in a fully protonated protein–ligand complex,
protein atoms should be listed as ATOM records, while ligand atoms should be listed as HETATM
records. Further, ensure that cofactors are given the correct heading in the PDB file (i.e., static metal
ions should be given ATOM records if they are to be included in the scoring grids).

bash $DOCKBASE/proteins/protein_prep/prep.sh protonated_input.pdb $PWD

The outputs are new rec.pdb and xtal-lig.pdb files, as well as a rec.crg.pdb file inside
a working directory. HID, HIE and HIP naming is generated automatically from the protonation
state of the HIS residues and again should be checked for accuracy.
Note: this script requires a path to the Chimera binary file and may need to be modified by the user.
9 Generate scoring grids and matching spheres from the new files (rec.pdb, xtal-lig.pdb, and
working/rec.crg.pdb), and examine the outputs as in Step 6:

$DOCKBASE/proteins/blastermaster/blastermaster.py --addNOhydrogensflag –v

Checking the files

10 Check that all files are generated. At the end of a successful blastermaster run, the directory should
contain an INDOCK file and two directories: working and dockfiles. The working directory
contains all the intermediate files used for grid generation. The dockfiles directory contains the
scoring grid files and matching spheres file. The INDOCK file contains all parameters to control the
docking program, such as sampling options and location of input files, i.e., docking grids and 3D ligand
conformer libraries (see the INDOCK Guide in Supplementary Information). In our experience, the
automated grid generation with blastermaster.py successfully produces reliable docking
parameters; however, nonstandard amino acids or particular atom types require additional information
and adjustments of force field parameters. Instructions on how to check and adopt the grid generation
are provided in the Troubleshooting section below and in the Blastermaster Guide (Supplementary
Information). For the provided example files generated for MT1, the protein–ligand complex was
modeled using the automated preparation pipeline in Maestro, Schrodinger48.

Section 2: collect and build the control ligand set ● Timing Minutes to hours for ligand
building, depending on the number of molecules
11 Collect the known actives (positive controls) for retrospective analysis. For a given target, these can
be found in the scientific literature, patent literature or public databases such as IUPHAR/BPS114,
ChEMBL115 or ZINC9,99,116, or available in-house.

Curate the actives list

CRITICAL While it may be possible to find dozens of actives, it is likely that many come from the
c

same chemical series. For a rigorous control analysis, redundant (i.e., highly similar) compounds should
be clustered and the most potent compound selected.
12 Sort all knowns by potency.
13 Cluster these based on 2D similarity using any preferred method. We suggest calculating clusters
based on ECFP4 Tanimoto similarities with a cutoff of 0.35.

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14 Use the most potent compound from each cluster as the representative of that scaffold. It is best to
refine these actives to a set that is representative of the chemical space intended for prospective
screening (i.e., with limits on molecular weight) so that the retrospective analysis will match the
prospective aims.
15 Find out whether the known ligands of the target are neutral or charged as one criteria of the
docking parameter calibration is the ability to score ligands with corresponding charges well.
16 Save a final list of actives’ SMILES as ligands.smi. Typically, 10–30 diverse actives represent a
good control set. For targets with less than this value, the controls are still useful but may not be as
informative. For the example of MT1 we extracted a set of 28 agonist and antagonists from the
IUPHAR/BPS database13.

Build the 3D conformer library of the actives

17 Go to the ‘build3d’ application on tldr.docking.org79.
18 Add the ligands.smi file to the ‘Input’ section.
19 Select ‘db2’ for the file type.
20 Click ‘Go’ to submit.
21 Download the build3d_results.tar.gz file, and move it to your work directory.
22 Decompress the tar file:

tar xvfz build3d_results.tar.gz

23 The results are in a build3d_results/ directory with two subdirectories failed/ and
finished/ indicating the build status of the compounds. In example data provided, all ligands
were built to completion.
24 To build a split database index file, or path file to the ligands, use the following command:

ls -d $PWD/build3d_results/finished/*.db2.gz > actives.sdi

25 To generate a list of actives’ IDs:

ls build3d_results/finished/*0.db2.gz | awk -F"/" '{print $NF}' | awk

-F"_" '{print $1}' > actives.id

Curate and build the property-matched decoy set

26 Go to the ‘dudez’ application on tldr.docking.org78.
27 Submit the ligands.smi file to the input section.
28 Click ‘Go’ to start the calculation. The ZINC database will be scanned for compounds that match
the following six properties of the input ligands: molecular weight, LogP, charge, number of
rotatable bonds, number of hydrogen bond donors, and number of hydrogen bond acceptors. Each
compound will then be compared with the actives for 2D similarity, with similar compounds being
discarded. A final set of 50 decoys per input active will be calculated.
29 Download the decoys_dudez.tar.gz file, and move it to your work directory.
30 Extract the files:

tar xvfz decoys_dudez.tar.gz

The files will live in a new directory new_decoys.

31 To obtain the split database index file of the decoys:

ls -d $PWD/newdecoys/decoys/*.db2.gz > decoys.sdi

32 To obtain a list of all the decoys’ IDs:

awk ‘{print $2}’ newdecoys/decoys.smi > decoys.id

33 Collect output files. At this point, four files should be generated: actives.sdi, actives.id,
decoys.sdi and decoys.id.

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Section 3: run retrospective docking calculations to test the binding pocket parameters
● Timing Minutes to hours depending on number of molecules and compute cores
34 In the directory where blastermaster was run (i.e., contains the INDOCK file and dockfiles
directory), copy over the ligand and decoys .sdi files and .id files.
35 Combine the two .sdi files:

cat actives.sdi ligands.sdi » controls.sdi

36 Check values in the INDOCK file

● Set the atom_maximum to 100. This value is a hard cutoff such that if a ligand has more than

this number of heavy atoms it will not be docked. For retrospective calculations, we want all
ligands to be docked and scored regardless of size, so we use a large value here
● Set the mol2_maximum_cutoff to 100. This value is a cutoff for saving 3D coordinates of

docked poses. If a ligand scores worse (i.e., more positive) than this cutoff, the pose will not be
saved. Setting a low value is useful in large-scale prospective screens to save on disk space and
computation time, but for retrospective screens we want all information saved for analysis
37 Set up the docking directory

$DOCKBASE/docking/setup/setup_db2_zinc15_file_number.py ./ controls
controls.sdi 1 count

This script will separate the controls.sdi file into one directory called controls0000. For
this first calculation, the size of the sdi file is manageable on a single core, though splitting it will be
faster (i.e., 10). For control calculations with 10,000–100,000 ligands and for large-scale prospective
screens, the sdi file should be split into multiple directories and the jobs distributed over multiple
cores. A dirlist file is generated that lists all of the split directories prepared
for docking.
38 Dock the control set. At this point, it is possible to submit the data to a computer cluster or do the
calculation on a single computer.
(A) Docking without submitting to a cluster
(i) Move into the controls0000 directory. You will find INDOCK (pointing to the
dockfiles/ directory) and a split_database_index file. These are all the inputs
that the DOCK program needs to run the calculation.
(ii) Run the docking calculation:

$DOCKBASE/docking/DOCK/bin/dock.csh

? TROUBLESHOOTING
(iii) The output files from the docking program are OUTDOCK, listing docking scores of
successfully docked molecules, computational performance or potential error messages,
and a zipped mol2 file (test.mol2.gz) containing the 3D poses of docked molecules.
(iv) Move back into the directory that contains dirlist when done.
(B) Docking with submitting to a cluster
(i) In the directory that contains the dirlist (created in the previous step), run one of the
following commands depending on cluster architecture:
● SGE: $DOCKBASE/docking/submit/submit.csh

● Slurm: $DOCKBASE/docking/submit/submit_slurm.csh

● PBS: $DOCKBASE/docking/submit/submit_pbs.csh

CRITICAL STEP If you use another cluster scheduler, adopt these scripts to your needs.
c

39 When the job has completed, the last line of the OUTDOCK file should contain an elapsed time. If
this is not the last line of the OUTDOCK file, an error has occurred (see Troubleshooting) and the
following script will not run.
? TROUBLESHOOTING
40 Extract the scores of all docked compounds

python $DOCKBASE/analysis/extract_all_blazing_fast.py ./ dirlist

extract_all.txt 100

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 PROTOCOL NATURE PROTOCOLS

The arguments are the dirlist, the name of the file to be written (extract_all.txt) and
the max energy to be kept (this value should match the mol2_maximum_cutoff value in the
INDOCK file). The important output file for future analysis is the extract_all.sort.uniq.
txt file, containing the rank-ordered list of docked compounds with only the highest score for
each compound.
41 Get the poses of the docked compounds

python $DOCKBASE/analysis/getposes_blazing_faster.py ./ extract_all.

sort.uniq.txt 10000 poses.mol2 test.mol2.gz

The arguments are the path where the docking is located (./), the name of the extract_all.
sort.uniq.txt file, the number of poses to get (10000, i.e., set to larger than the number of
compounds docked for retrospective calculations since we want to get all), the name of the output
mol2 file (poses.mol2), and the name of the input mol2 files (test.mol2.gz), containing
3D coordinates of predicted poses from the docking calculation (located in the
controls0000 directory).
42 Get the poses of just the actives

python $DOCKBASE/analysis/collect_mol2.py actives.id poses.mol2

actives.mol2

The arguments are the file containing active IDs, the pose file containing all of the compounds both
actives and decoys, and the name of the output file to be written.
43 Calculate the LogAUC early enrichment

python $DOCKBASE/analysis/enrich.py -i . -l actives.id -d decoys.id

Inputs are the working directory (‘.’ if in the directory where the extract_all file is located) and
the two files containing the IDs of actives and decoys. The output is a roc.txt and roc_own.
txt file. Both files report an AUC and LogAUC and contain the grid points for plotting receiver
operator curve (ROC) plots. The roc.txt file is calculated over all inputs in the ID files, and the
roc_own.txt file is calculated only over the compounds that successfully docked.
44 To generate a plot (roc_own.png) of the roc_own.txt file:

python $DOCKBASE/analysis/plots.py -i . -l actives.id -d decoys.id

45 Calculate the charge distribution by DOCK score

python $DOCKBASE/analysis/get_charges_from_poses.py poses.mol2

charges

Inputs are the poses files and the name of the output file to be written. The output charges file is
a list of DOCK scores and the charge of the ligand with that score.
46 To generate a plot of total DOCK score by ligand charge (charge_distributions_vs_
energy.png) run:

python $DOCKBASE/analysis/plot_charge_distribution.py charges

47 Plot the contribution of each score term in the energy function outlined in Equation 1
(energy_distributions.png):

python $DOCKBASE/analysis/plot_energy_distributions.py extract_all.

sort.uniq.txt

48 Collate the output files. The important outputs of these steps are: actives.mol2, roc_own.
png, energy_distributions.png and charge_distributions_vs_energy.png.

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 NATURE PROTOCOLS PROTOCOL
a Key interactions of between docked b
molecules and MT1 Enrichment of actives against decoys
100
LogAUC
Default DUDE-Z: 1.89
Optimized DUDE-Z: 10.35
Default extrema: 17.86
Asn162 75
Optimized extrema: 25.66

%ligands found
Gln181
50

25

0
10–1 100 101 102
%decoys found (log)

c Energy distribution breakdown d Charge distribution

20 Default DUDE-Z –10

Optimized DUDE-Z

–20

–30

Score
Score

–20 –40

–50
Default extrema
Optimized extrema
–40

Electrostatics Van Ligand –1 0 1

der waals desolvation Charge

Fig. 6 | Controls for docking optimization. a, The receptor (blue) is shown with the crystallized ligand (orange).
Docked control actives are shown in green and yield similar poses and interactions as the crystal ligand. The two
residues, Asn162 and Gln181, that have their dipoles artificially increased (‘polarized’) to enhance the weight of polar
interactions are shown hydrogen bonding to the crystal ligand. b, A log-transformed ROC plot is shown comparing
the rate of identifying ligands versus decoys. A random selection would follow the dashed black line. The area under
this dashed line is subtracted from the values reported for LogAUC such that a curve above the line would have a
positive LogAUC, a curve below the line would have a negative LogAUC, and a curve following the dashed line would
yield a LogAUC value of zero. Shown are the curves for the default settings and optimized settings for either the
DUDE-Z control set and the Extrema control set. In both cases, the overall LogAUC value increases and the early
enrichment improves. c, The energy distribution breakdown shows the individual score terms for each scored
molecule in the docked setup. Based on this breakdown, it is clear that VDW interactions primarily drive ligand
recognition. However, in the optimized setup in which electrostatic spheres with a radius of 1.9 that extend the
dielectric boundary are used, the electrostatic score term shifts to more negative values. The desolvation spheres at
the dielectric boundary in the optimized setup, with a radius of 0.1, have only minor effects on the ligand desolvation
score term. d, In the Extrema challenge, the top-ranking ligands are plotted by their charge and DOCK score. In the
Default settings, there is a preference for neutral ligands followed closely by monocations. The Optimized settings
enhance the preference for neutrals.

Evaluate the docked poses of the actives

49 Open the rec.pdb and xtal-lig.pdb in Chimera, and prepare the visualization of the
binding pocket to the user’s preference.
50 In the Tools tab, select Structure/Binding Analysis and click ViewDock.
51 In the menu, navigate to your directory and open actives.mol2.
52 For file type, select any of the DOCK options. This will open a window (ViewDock) to navigate
through all of the molecules in this file such as in Fig. 6a.

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53 Under the ‘Column’ tab, several details about the docked ligands can be listed, e.g., Total Energy,
Electrostatic or Van der Waals terms.
54 In the Tools tab, under Structure/Binding Analysis, ‘Find HBonds’ can be used to identify hydrogen
bonds between the protein and ligand molecules.
55 Evaluate the docked poses, asking the following questions:
● Do the ligands occupy the part of the pocket as expected?

● Do they make the types of interactions anticipated from what is known about the ligands and

the pocket?
● Are there aberrant or unsatisfied interactions? For example, in MT , it is known from the crystal
1
structure that ligands can form hydrogen bonds with Asn162 and Gln181. Issues with binding
poses are usually a sampling problem, but scoring terms such as electrostatics can influence
specific interactions
● If many actives do not dock, is there a reasonable explanation (e.g., selected controls are larger than

the pocket volume as has been observed when attempting to dock antagonists into agonist pockets)?
Optimization of sampling is performed with a matching sphere scan as detailed in Steps 59–64

Evaluate the enrichment and scoring metrics

56 Evaluate the docking parameters’ ability to discriminate actives from decoys. The roc_own.png
file shows the plot of the rate of finding actives as a function of decoys. The AUC is indicative of
this discriminatory power (Fig. 6b). A positive LogAUC value is a sign that actives are enriched over
decoys, a value near 0 represents random selection, and a negative value demonstrates that the
model prefers decoys over actives. This may be a result of poor poses, improper scoring or both. It
is best to optimize poses before pushing forward on scoring discrimination. Even for good LogAUC
values, it is important to evaluate the poses as in Step 55 as it is possible to get good scoring actives
that do not dock in the correct pose.
57 Examine the energy contributions of the docked poses. In the energy_distribution.png file
(Fig. 6c), the total DOCK scores for the docked poses are broken down into the main components
of the scoring function: electrostatics, VDW and polar ligand desolvation.
Do the contributions of the various score terms match the properties of the binding pocket? In
the MT1 pocket, the VDW interactions dominate because of its largely hydrophobic nature. For
targets forming salt bridges to ligands, electrostatic terms should at least be balanced with VDW
scores. If the balance does not match with what is expected for the pocket, the strength of the
scoring terms can be modified in Steps 65–68.
58 Evaluate the charge preference of the docking parameters. If the electrostatics term dominates in
Step 57, the scoring function will likely prefer highly charged ligands. This can be measured in the
charge_distributions_vs_energy.png plot (Fig. 6d), which shows the charge state of
the top scoring molecules. DUDE-Z decoys should have charges matching the active ligands.
However, in property-unmatched decoys such as the Extrema set78 (Steps 78–88) and in-stock set
(Steps 89–97), it is important to ensure the docking is not biased toward charge extremes, which
can suggest an overweighting of electrostatic terms; this can be addressed in Steps 65–68.

Section 4: optimize poses by modifying matching spheres ● Timing Minutes to build; ≤1 h

to dock
59 Create a new directory matching_sphere_scan/ that contains the INDOCK file and
working/ and dockfiles/ directory from the previous directory.
60 Run the following script to create sets of matching spheres in which the matching spheres that do
not map to atoms in the original xtal-lig.pdb file are perturbed as in Fig. 7a: (~1 min)

python $DOCKBASE/proteins/optimization/scramble-matching-spheres.py
-i $PWD/dockfiles -o $PWD -s 0.5 -n 50

The arguments are: -i, the path to the dockfiles/ directory; -o, the path to where new docking
directories should be created; -s, the maximum distance to move a matching sphere
(0.5 recommended); -n, the number of perturbed sphere combinations to create. For a single
receptor target, we recommend 50–100 sets of sphere sets.
61 Each new directory should contain an INDOCK file and dockfiles/ directory. Check that the
matching_spheres.sph file in the dockfiles/ directory is unique to each new directory created.

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 NATURE PROTOCOLS PROTOCOL
a b

Fig. 7 | Matching and dielectric boundary spheres drive changes to sampling and scoring in DOCK3.7. a, the crystal
ligand is shown as orange sticks in the receptor pocket (gray). Matching spheres derived from the coordinates of the
crystal ligand are shown in yellow and remain fixed during sphere perturbation. Random spheres (blue) are
calculated with the program SphGen, and a set of spheres are selected that are near the crystal ligand. In a matching
sphere scan, only the random spheres are perturbed and a new set is obtained (green). b, the crystal ligand (orange)
is again shown in the context of the receptor binding pocket (gray). Dielectric boundary spheres (cyan) cover the
binding surface around the crystal ligand to alter the electrostatic or desolvation potentials at the boundary between
solvent and protein.

62 For each directory, copy over the controls.sdi file.

63 Run the docking calculation and extract the poses as in Steps 37–48.
64 Examine the new actives.mol2 files and LogAUC values from the different matching sphere
sets. Ideally, a set of spheres will increase the LogAUC and improve the binding poses of the actives.
For MT1, the LogAUC increased from ~2 to 5. While not a large change, there was improved
placement of the flexible components of the active ligands, which suggests better sampling and pose
identification in a prospective screen.
If the poses did not improve during this matching sphere scan, it may indicate that there is a
problem with the binding pocket model, the ligand set, the sampling of ligand conformations, or
the placement of the crystallographic matching spheres. Examining each of these can help improve
the binding poses in this first control calculation. Alternatively, the scoring function needs to be
optimized to score the expected pose better (see Steps 65–68).
? TROUBLESHOOTING

Section 5: optimize ligand scoring by modulating the protein–water dielectric interface

(adding a layer of dielectric boundary spheres) ● Timing 15 min on cluster; 20–30 min per
radius on local machine
65 Create a new directory with the INDOCK file and working/ and dockfiles/ directories from
the best matching sphere set (or the default settings if no matching sphere scan was run).
66 Generate the boundary modifying spheres (Fig. 7b)

$DOCKBASE/proteins/optimization/boundary-sphere-scan.sh -b $PWD -v

This version of the script will run on an SGE cluster. Modify the submission script and scheduler as
needed for different cluster architecture. To run on a local machine, use the script:

$DOCKBASE/proteins/optimization/boundary-sphere-scan_no_cluster.sh
-b $PWD -v

67 Combine the different radii boundary spheres (5 min):

python $DOCKBASE/proteins/optimization/combine-grids.py -b $PWD

This script generates a set of combinations of boundary modifying spheres across both ligand
desolvation and low dielectric spheres. These directories have everything needed to run individual

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 PROTOCOL NATURE PROTOCOLS

N ASN –0.520 8 ASPARAGINE N ASM –0.520 8 ASPARAGINE polarized

CA ASN 0.217 4 CA ASM 0.217 4
C ASN 0.526 1 C ASM 0.526 1
0 ASN –0.500 11 0 ASM –0.500 11
CB ASN 0.003 3 CB ASM 0.003 3
ND2 ASN –0.867 8 ND2 ASM –0.867 8
CG ASN 0.675 1 CG ASM 0.675 1
0D1 ASN –0.470 11 0D1 ASM –0.870 11
H ASN 0.248 6 H ASM 0.248 6
HD21 ASN 0.344 6 HD21 ASM 0.544 6
HD22 ASN 0.344 6 HD22 ASM 0.544 6
CA CA

CB CB

CG CG HD21
HD21
0D1
ND2 ND2
0D1

HD22
HD22

Fig. 8 | Polarizing effects specific atoms’ electrostatic potential. In contrast to global modifications to the electrostatic potential with the
incorporation of thin spheres, polarizing allows for very specific modifications to a residue’s charge status. A canonical asparagine (ASN) from the
prot.table.ambcrg.ambH file is shown with its polarized version ASM in which the carbonyl becomes more electronegative while the amide
hydrogens become more electropositive to maintain the overall charge. The electrostatic potential corresponding to each atom is shown as spheres,
with red corresponding to negative charge and blue corresponding to positive charge.

retrospective calculations except for the controls.sdi file that will need to be copied into each
one. Repeat the retrospective calculations from Steps 37–48.
68 Following retrospective calculations across the combinations of dielectric boundary sphere radii,
select a combination that ideally improves LogAUC (Fig. 6b) and helps to balance the score
distributions in the energy_distribution.png plot (Fig. 6c). If there are a number of
equally performing combinations, subsequent control calculations can help identify the best set for
your system (see Steps 78–97). In the case of MT1, we chose electrostatic spheres of radius 1.9 and
desolvation spheres of radius 0.1 as they increased LogAUC and enhanced the charge distribution
of compounds to favor neutrals to match known MT1 ligands (Fig. 6d).

Section 6: (optional): polarize (or depolarize) residues to effect electrostatics

● Timing 15 min
CRITICAL If after these scans a particular interaction is still missed or erroneously captured, it may be
c

necessary to modify the partial charges of a particular residue. For example, in the case of MT1, the
docking scores are largely dominated by VDW interactions (Fig. 6c). However, two residues Gln181 and
Asn162 form hydrogen bonds with the known ligands. For these specific interactions, a global
modification to the dielectric boundary (Steps 65–68) may not be sufficient and, instead, a local
modification to partial charges as outlined in Steps 69–77 may enhance favorable scores for these
interactions. For targets where this is not necessary, proceed to Step 78.
69 Make a new directory for polarizing residues. Copy over rec.pdb, xtal-lig.pdb, working/
rec.crg.pdb, working/amb.crg.oxt and working/prot.table.ambcrg.ambH
from the previous docking setup.
70 In the rec.crg.pdb file, rename the residues to be polarized with a unique three-letter amino
acid code. For example, in the MT1 receptor, we chose to polarize GLN181 and ASN162 to
enhance the polar interactions between actives and these hydrogen bonding residues. Accordingly,
we renamed GLN181 as GLD and ASN162 as ASM (see Fig. 8).

sed -i 's/GLN A 181/GLD A 181/g' rec.crg.pdb

sed -i 's/ASN A 162/ASM A 162/g' rec.crg.pdb

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 NATURE PROTOCOLS PROTOCOL
Generate partial charges for the new residues
CRITICAL The two files that read in partial charges are the amb.crg.oxt and prot.table.

c
ambcrg.ambH files located withing the working/ directory. These files contain default partial
charges for all amino acids and some special residues such as ions. Make the following changes in both
amb.crg.oxt and prot.table.ambcrg.ambH files.
71 Create new sections for the polarized residues in the two files by copying the standard residue
partial charges.
72 Rename the polarized residues to match the three-letter codes used in the rec.crg.pdb file. The
names are all capitalized in the prot.table.ambcrg.ambH file as shown in Fig. 8 and all
lowercase in the amb.crg.oxt file.
73 Redistribute the charge around the atom of interest, making sure that the net charge remains the
same. We suggest testing modifications of 0.2 or 0.4 charge units for a given atom. For example, for
an ASN-to-ASM change, where we want to enhance the electronegativity of the sidechain carbonyl
(OD1), increase the charge by 0.4 units, resulting in a change from −0.470 to −0.870. As −0.4
charge was added to the residue, +0.4 must be distributed to other atoms in the residue to maintain
the net charge. One option is to add +0.2 to each of the sidechain amide hydrogens (HD21 and
HD22) as in Fig. 8, though the charge could have been distributed to the backbone amide or
another atom not involved in binding.

Running the grid generation

74 Before running the grid generation, check that the following files are present: rec.pdb, xtal-
lig.pdb, the modified amb.crg.oxt and prot.table.ambcrg.ambH files, and a
working/ directory with the modified rec.crg.pdb file.
75 Run blastermaster with the following command:

$DOCKBASE/proteins/blastermaster/blastermaster.py --addNOhydrogensflag
--chargeFile=amb.crg.oxt --vdwprottable=prot.table.ambcrg.ambH –v

? TROUBLESHOOTING
76 Rerun control calculations as in Step 37–48. When examining the new poses, consider the following questions:
● Does the polarized residue promote or discourage the desired interaction?

● Are the new interactions scored more favorably?

77 Rerun the dielectric boundary modifying sphere scan in Steps 65–68. We recommend doing this
because the new polarized residue(s) will have altered the electrostatics grid. It may be necessary to
test different combinations and strengths (0.2, 0.4) of the polarized residues to identify the best-
performing set of parameters that enhances correct interactions, improves poses and ideally
improves LogAUC. However, due to the modification to the electrostatic potential introduced by
these changes, it is important to ensure these improvements do not come at the cost of biasing the
screen towards overly charged molecules. This bias can be checked in the next section Steps 78–88.

Section 7: Extrema charge control calculations ● Timing 2–3 h for compound building
Generate the Extrema set
CRITICAL Compounds in the extrema set come from the area of chemical space occupied by the
c

known actives (molecular weight and LogP) but distinct charges (−2, −1, 0, +1, +2). These decoys will
be used to measure biases introduced into the scoring function owing to modifications to the
electrostatic potential.
78 On tldr.docking.org, navigate to the ‘extrema’ application.
79 Upload the SMILES of your active ligands used in section II to the webpage.
80 Select 500 or 1,000 molecules per charge, and click the ‘Go’ button.
81 After a few minutes, a results file will be available to download and extract. Within the file should be
five SMILES files grouped by charge. Extract the SMILES file from the download:

unzip extrema_results.zip

82 Build the extrema set.

83 For each SMILES file, submit the file to the ‘build3d’ application on tldr.docking.org. Depending on the
number of ligands in each file, and the activity on the website, each file should take 2–3 h to build.

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 PROTOCOL NATURE PROTOCOLS

84 When all sets are built and downloaded, combine the path to all compounds in a split database
index file along with the known actives.

Run the extrema set

85 Navigate to a directory containing the INDOCK file and dockfiles/ directory that you want to
test. The dockfiles/ directory should contain the set of matching spheres and the dielectric
boundary sphere combination selected from the previous steps.
86 Prepare the docking run. In the previous steps, the control ligand set was small enough to run in a
single run. This control set often has 10,000+ compounds, and it is best to split this into multiple
jobs. To do this run:

$DOCKBASE/docking/setup/setup_zinc15_file_number.py ./ extrema
extrema.sdi 100 count

87 Submit the docking calculations over a cluster as in Step 38B.

88 Repeat the analysis in Steps 39–48 with a focus on the charge_distributions_vs_
energy.png plot (Fig. 6d). An optimal set of docking parameters will have good LogAUC with early
enrichment, and the top-ranking compounds will contain charges that match the charges of the known
actives. Sometimes multiple boundary-modifying sphere combinations will be tested in this Extrema
calculation to find a set that enhances the scoring preference for compounds similar to knowns.

Section 8: run a ‘small’-scale in-stock screen ● Timing 1–24 h depending on

download speed
89 To collect an in-stock screening library, go to http://zinc20.docking.org/tranches/home/.
90 At the top of the tranche viewer (Fig. 9), select ‘3D’ representation, set Purchasability to ‘In-stock’,
and select the charge states of interest to your target (i.e., only 0). Next, select the molecular weight
range you want to screen. Finally, select a LogP range tailored to your target. If no specific LogP
range is desired, we recommend LogP ≤ 3.5 to avoid insoluble compounds that might aggregate and
yield false positives in the experimental assay.
91 When only the tranches of interest are selected, click the download button in the upper right corner
of the screen. Select ‘DOCK37’ as the download format and ‘cURL’ or ‘WGET’ as the download
method. When done, click ‘Download’ to obtain a text file with all the cURL or wget commands
needed to download the set of selected tranches.
92 Prepare a split database index file with the path to all of the newly downloaded compounds.
93 Split the docking run over 1,000 jobs to efficiently run the screen as the number of compounds is
usually ~1–4 million.

$DOCKBASE/docking/setup/setup_zinc15_file_number.py ./ instock
instock.sdi 1000 count

94 Run the screen on a cluster as in Step 38B.

95 Run the analysis of the screen to generate a LogAUC. It should be on par or better than LogAUC
values seen previously, as the set of decoy molecules grows in size and departs further from the set
of known ligands.
96 Test out hit picking strategies. Use any metric for filtering hits (Table 2), and visually examine the
hits that pass the selected filters.
● Are the compounds forming the expected interactions with the binding pocket?

● Are they sampling in the correct region of the pocket?

If there are any issues, determine if it is a sampling or scoring problem and alter the previous steps
(matching spheres or dielectric boundary modifying spheres, respectively) to optimize. If the top
poses capture expected interactions, then it is worth moving into a large-scale prospective screen.
97 Choose settings for the large-scale docking experiment. If multiple docking parameters were tested
in this section (i.e., section 8), one method for selecting the best settings for large-scale docking is to
choose the parameters that yield the greatest number of viable compounds after all of the post-
docking filters have been applied. This will ensure a large number of hits in the large-scale
campaign. Alternatively, one can purchase hits from the different screens and see which setup leads
to more true actives in an experimental setting. Even if just a few low-potency hits are obtained at
this point, it may suggest which interactions are critical for activity in the pocket.

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 NATURE PROTOCOLS PROTOCOL
ZINC20 tranche viewer
http://zinc20.docking.org/tranches/home/ In-stock: on vendor
shelves Ability to
Wait ok: ready to (de)select
make different ligand
charges

2D: all annotated

compounds
3D: all prebuilt
compounds

Toggle buttons to
select/deselect
columns/rows

cURL and WGET

commands for
downloading
compound library

Total count of
selected compounds

Fig. 9 | Navigating the ZINC20 Tranche Viewer. Several options are available at http://zinc20.docking.org/tranches/home/ for selecting different
subsets of ligands for virtual screening. Important criteria such as selecting between 2D/3D, purchasability, charge, molecular weight and logP are
highlighted. To download compounds, different methods such as downloading as an index file or directly downloading with cURL and
WGET are shown.

Section 9: large-scale docking ● Timing 1–5 d, depending on number of compounds and

compute nodes
98 Create a new directory for the large-scale campaign with the final set of parameters, i.e., the
INDOCK file and the dockfiles/ directory.
99 Change the mol2_maximum_cutoff in the INDOCK file to a value likely to eliminate ~90% of
the docked molecules to save on disk storage as compounds with total scores worse than a certain
value (more positive) are unlikely to be considered hits from the large screen and should not be
saved (use the results of the in-stock screen to help inform on the best mol2_maximum_cutoff
values to use).
100 Obtain the ZINC20 library. The ZINC20 library contains a prebuilt 3D conformer library of
nearly 700 million fully protonated and tautomerized compounds (i.e., protomers). The library
is largely built on Enamine’s readily accessible library, which consists of molecules that have
a >80% likelihood of synthesis in one or two reaction steps11. This library can be downloaded
from https://files.docking.org/3D/ or using the tranche browser as in Fig. 9 and consists of
~60 TB of data.
101 Select properties of the ZINC20 library for the screen such as molecular weight, charge and LogP.
We recommend screening compounds separately by MWT range (i.e., fragments ≤ 250 amu) as
larger compounds often score better due to their ability to form additional interactions within the

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binding pocket117. To obtain a split database index (sdi) file for a predownloaded ZINC20 database,
use the ‘DOCK Database Index’ download format in the tranche viewer (Fig. 9) and provide a path
to the downloaded database in the ‘ZINC DB Root’ field.
102 Split the docking campaign over 1,000 or more jobs. Typically, ~100 million compounds can be
screened per day on an academic 1,000 core cluster of recent vintage (as of 2020).

$DOCKBASE/docking/setup/setup_zinc15_file_number.py ./ largescale
docking zinc20library.sdi 1000 count

103 Run the screen on a cluster as in Step 38B.

Section 10: hit picking ● Timing Days to weeks

104 Extract the scores of the top compounds as in Step 40 with the maximum score cutoff set to the
mol2_maximum_cutoff set in Step 99

python $DOCKBASE/analysis/extract_all_blazing_fast.py ./dirlist

extract_all.txt -50

105 Collect between 300,000 and 1,000,000 poses from the best-scoring compounds.

python $DOCKBASE/analysis/getposes_blazing_faster.py ./ extract_all.

sort.uniq.txt 1000000 poses.mol2 test.mol2.gz

106 Use any number of post-docking filters including but not limited to those described in Table 2.
107 From the compounds that remain after filtering, visually examine up to 5,000 molecules depending
on how viable the docked compounds look. We recommend that visual examination is done by
more than one person, because of the different knowledge base that each individual brings to the
selection process (i.e., a medicinal chemist will prioritize different features over a molecular
biologist and vice versa)19. This step should be done before prioritizing compounds for purchase, as
we have found human-picked compounds have better efficacy than compounds obtained from fully
automated hit-picking12. By using post-docking filters as in Step 106 prior to visual examination,
one can search more deeply through the list of top ranked molecules to identify molecules
for testing.
108 Choose 50–200 compounds for wet-lab testing depending on price, capability of testing and
confidence of success as informed from retrospective calculations.

Troubleshooting
Structure preparation
Alternative conformations or missing side chains should be completely modeled before starting the
grid generation (blastermaster.py). Otherwise, any superfluous or missing atoms will result in
erroneous VDW surface or partial charge calculations (see below).

Blastermaster
Section 1, Step 5
All input and output files as well as options and flags of the blastermaster program are listed in the
Blastermaster Guide (see Supplementary Information). Of important note is that blastermaster
requires protein and ligand structures to be provided as PDB files called rec.pdb (and working/
rec.crg.pdb in case a protonated structure is used) and xtal-lig.pdb. Alternative file names
will not be recognized in the default settings.

Section 1, Step 10
If blastermaster does not successfully finish the grid generation, log files in the working directory
will yield the required information to backtrace the error (see Blastermaster Guide in Supplementary
Information). We suggest running blastermaster in the verbose mode (-v) as it allows to easily detect
at which step the program failed.

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Most common errors are related to unknown atom or residue types in rec.pdb (and/or rec.
crg.pdb). Missing parameters for specific atom types can be added to the following parameter files:

$DOCKBASE/proteins/defaults/radii (required for surface calculation by

the dms program)
$DOCKBASE/proteins/defaults/amb.crg.oxt (partial charges for qnifft)
$DOCKBASE/proteins/defaults/vdw.siz (Van der Waals radii for qnifft)
$DOCKBASE/proteins/defaults/prot.table.ambcrg.ambH (atom typing for
chemgrid)
$DOCKBASE/proteins/defaults/vdw.parms.amb.mindock (Van der Waals
parameters for minimizer)

In our experience, most protein input structures can be converted into complete docking grids with
the default parameters provided in DOCKBASE. For some modifications such as capped termini or
structural waters, parameters are included in the provided files (e.g., amb.crg.oxt) but may use
slightly different atom names compared with default names from modeling programs such as Chi-
mera, PyMol or Maestro. In these cases, we suggest adapting the corresponding atom names in the
input protein structure files (rec.pdb, rec.crg.pdb) rather than adding more atom types to
the default parameter files. Common naming errors occur for disulfide bonded cysteines (CYX in
prot.table.ambcrg.ambH) and water atoms (HOH, TIP, WAT and SPC in prot.table.
ambcrg.ambH).
The qnifft program may crash if too many atoms (>50,000) are present in rec.crg.pdb. While
we do rarely encounter this problem, particularly large proteins or other molecular complexes may
have to be reduced in atom number (e.g., removing fusion proteins or distal monomers). Typically,
protein segments >20 Å away from the binding pocket are unlikely to influence the resulting
docking grids.
In certain cases, blastermaster may be able to calculate all grids, even though some parameters
might be missing. For example, if nonstandard amino acids are used, the protonation state as well as
partial charges or VDW surfaces may not be computed correctly, but will not cause blastermaster
to crash.
To check if all force field parameters were assigned to the protein structure correctly, we suggest
visual inspection of docking grids (Chimera). The scoring grids (vdw.vdw, trim.electro-
statics.phi, as well as ligand.desolv.heavy and ligand.desolv.hydrogen) are
found in the dockfiles directory. The following scripts can be run in the dockfiles directory to
convert the grids into dx files for visualization.
Convert VDW grid:

python $DOCKBASE/proteins/grid_visualization/create_VDW_DX.py

Input: vdw.vdw, vdw.bump (default)

Output: vdw.dx (default)
Convert electrostatics grid:

python $DOCKBASE/proteins/grid_visualization/create_ES_DX.py trim.

electrostatics.phi trim.electrostatics.dx

Input: trim.electrostatics.phi
Output: trim.electrostatics.dx
Convert desolvation grid:

python $DOCKBASE/proteins/grid_visualization/create_LigDeSolv_DX.py

Input: ligand.desolv.heavy (default)

Input: ligdesolv.dx (default)

To visualize the grids, first open the rec.crg.pdb file in Chimera. The dx files can be opened with
the Volume Viewer application. The vdw.dx file should resemble the surface of the receptor. The
ligdesolv.dx file demonstrates various solvation levels of the pocket (the grid itself is a

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continuum). By changing the level of the representation, the volume should fill the pocket illustrating
the ligand moving from a less solvated state to the most solvated state. For the trim.electro-
statics.dx file, it is best to set the minimum and maximum levels to −100 and 100, respectively.
Then, set the colors to red for −100 and blue for 100. This now shows regions of positive partial
charges (blue) and negative partial charges (red). For all grids, ensure that the representations match
what is expected for the binding pocket and that there are no regions of missing grid points. If there
are obvious holes in any of the grids, in close proximity to the ligand, certain residue types were not
identified correctly and corresponding parameters could not be assigned.
A list of all partial charges assigned to rec.crg.pdb is given in working/qnifft.atm
(column 11). If the total charge of the system does not sum up to an integer value, certain partial
charges may not have been found in $DOCKBASE/proteins/defaults/amb.crg.oxt.
Furthermore, if many partial charges for a given residue (or other structural components such as
metals or water molecules) are 0, proper parameters were likely not assigned. Specific errors in partial
charge assignment can be found in working/OUTPARM.

Docking
Section 3, Step 38A(ii)
The DOCK executable ($DOCKBASE/docking/DOCK/bin/dock64) will only read a file called
INDOCK.

Section 4, Step 64
If ligands dock in poses far from the binding pocket, visualize the matching spheres to ensure they
occupy the same area as the xtal-lig. The matching_spheres.sph file in the dockfiles
directory contains the set of matching spheres. To view this file in a protein visualization application,
they first must be converted to a pdb file.

csh $DOCKBASE/proteins/showsphere/doshowsph.csh matching_spheres.sph

1 matching_spheres.pdb

Input: matching_spheres.sph
Output: matching_spheres.pdb

The output file, matching_spheres.pdb, can be visualized with the receptor and ligand in either
Chimera or PyMOL. The matching spheres should align to the ligand’s heavy atoms, and additional
random spheres should be scattered around the ligand as in Fig. 7a.
If many ligands fail to dock (>50%), a number of issues may be at fault. It is common for 10–20%
of ligands to fail to dock in retrospective calculations because of mismatches between the pocket and
ligand properties. In the OUTDOCK file, two common statements may indicate these issues: ‘Grids
too small’ and ‘Skip size’. The ‘Grids too small’ statement indicates that the ligands do
not fit in the binding pocket. The ‘Skip size’ statement is used when the number of atoms in the
ligand exceeds the atom_maximum value in the INDOCK file. Adjusting this number to a higher
value will ensure that larger ligands will be docked and scored. If the OUTDOCK file shows many
compounds getting scored but the poses are not being saved, the mol2_maximum_cutoff value
may be too stringent. Relaxing this term will save more poses, but be careful to not go too high as the
size of the output files will correspondingly increase and may be an issue for disk storage capacity.
Additional INDOCK parameters can be adjusted according to the INDOCK Guide available in the
Supplementary Information.

Anticipated results
At the end of this protocol, a receptor will have been converted into a dock-readable binding pocket,
the system optimized on retrospective control calculations, and the system screened prospectively
against a large chemical library. While this protocol has been specific to a single target that yielded
successful results13, we have equally applied this protocol to a number of targets with general success
demonstrating its broad applicability12,83,118. We have attempted to curate all of the relevant steps,
though expert intuition brought by each user for their target will result in a different selection of final
hits for purchase. For these intuition-based steps, we recommend multiple people review the data
before making the final decision regarding which compounds to buy.

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 NATURE PROTOCOLS PROTOCOL
If none of the purchased compounds demonstrates activity at the target of interest as determined
experimentally, there remain several reasons for failure, including:
● The calculations were performed using the wrong conformation of the binding pocket

● The compound library did not contain enough examples of the class of compound most likely to dock

● Retrospective optimization was not possible due to lack of known chemical matter

● The experimental structure contained ambiguities inside chain or loop placement due to poor electron

density or refinement errors

● Incorrect or low-quality homology model

For example, targets that bind dianions or dications will suffer from much smaller library sizes,
while targets that bind large protein ligands may be difficult to modulate with small molecules. As in
experimental biology, no set of retrospective controls will ever guarantee prospective success, and the
many approximations in docking ensure that we still measure success by hit rates.
Nevertheless, these controls and optimization steps can reduce obvious sources of failure, and
allow one to better design a subsequent campaign. The experience of the field by now suggests that
the approach is likely enough to succeed to be worth the investment, while the novel ligands
that result can bring genuinely new biological insight to a field, a longstanding goal of the structure-
based enterprise.

Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary
linked to this article.

Data availability
An example set of files used in this protocol, including ligand and decoy sets, default docking grids and
optimized docking grids, can be downloaded from http://files.docking.org/dock/mt1_protocol.tar.gz. The
example dataset uses the MT1 structure (PDB: 6ME3) co-crystallized with 2-phenylmelatonin.

Software availability
DOCK3.7 can be downloaded after applying for a license from http://dock.docking.org/Online_
Licensing/index.htm. Licenses are free for nonprofit research.

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Acknowledgements
This work was supported by NIH grants R35GM122481 (to B.K.S.) and GM133836 (to J.J.I.). J.C. was supported by grants from the
Swedish Research Council (2017-04676) and the European Research Council (ERC) under the European Union’s Horizon 2020 research
and innovation programme (grant agreement: 715052). B.J.B. was partly supported by an NIH NRSA fellowship F32GM136062. C.M.W.
was partly supported by the National Institutes of Health Training Grant T32 GM007175, NSF GRFP and UCSF Discovery Fellowship.
We thank members of the Shoichet lab for feedback on the procedures described.

Author contributions
B.J.B. and S.G. wrote the manuscript with additional input from all authors. B.J.B., S.G., A.L., J.L., C.W., R.M.S., E.F., T.E.B., J.C., J.J.I. and
B.K.S. developed the protocol. B.J.B., S.G., A.L., J.L., C.W., R.M.S. and T.E.B. contributed scripts. E.F. tested the protocol. Research was
supervised by J.C., J.J.I. and B.K.S.

Competing interests
B.K.S. and J.J.I. are founders of Blue Dolphin Lead Discovery LLC, which undertakes fee-for-service ligand discovery.

Additional information
Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596-021-00597-z.
Correspondence and requests for materials should be addressed to Brian K. Shoichet.
Peer review information Nature Protocols thanks Claudio Cavasotto, Vincent Zoete and the other, anonymous reviewer(s) for their
contribution to the peer review of this work.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 20 January 2021; Accepted: 22 June 2021;

Published online: 24 September 2021

Related links
Key references using this protocol
Stein, R. M. et al. Nature 579, 609–614 (2020): https://doi.org/10.1038/s41586-020-2027-0
Lyu, J. et al. Nature 566, 224–229 (2019): https://www.nature.com/articles/s41586-019-0917-9
Schuller, M. et al. Sci. Adv. 7, eabf8711 (2021): https://advances.sciencemag.org/content/7/16/eabf8711

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 nature research | reporting summary
Corresponding author(s): Brian K Shoichet
Last updated by author(s): 06/09/2021

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