# Reference guide¶

## Preface¶

It is intended to develop this document into a full reference guide for the rDock platform, describing the software tools, parameter files, scoring functions, and search engines. The reader is encouraged to cross-reference the descriptions with the corresponding source code files to discover the finer implementation details.

## Acknowledgements¶

Third-party source code. Two third-party C++ libraries are included within the rDock source code, to provide support for specific numerical calculations. The source code for each library can be distributed freely without licensing restrictions and we are grateful to the respective authors for their contributions.

• Nelder-Mead’s Simplex search, from Prof. Virginia Torczon’s group, College of William and Mary, Department of Computer Science, VA.

• Template Numerical Toolkit, from Roldan Pozo, Mathematical and Computational Sciences Division, National Institute of Standards and Technology

## Introduction¶

The rDock platform is a suite of command-line tools for high-throughput docking and virtual screening. The programs and methods were developed and validated from 1998 to 2002 at RiboTargets (more recently, Vernalis) for proprietary use. The original program (RiboDock) was designed for high-throughput virtual screening of large ligand libraries against RNA targets, in particular the bacterial ribosome. Since 2002 the programs have been substantially rewritten and validated for docking of drug-like ligands to protein and RNA targets. A variety of experimental restraints can be incorporated into the docking calculation, in support of an integrated Structure-Based Drug Design process. In 2006, the software was licensed to the University of York for maintenance and distribution and, in 2012, Vernalis and the University of York agreed to release the program as open-source software.

rDock is licensed under GNU LGPL version 3.0 with support from the University of Barcelona - rdock.sourceforge.net.

## Configuration¶

Before launching rDock, make sure the following environment variables are defined. Precise details are likely to be site-specific.

• RBT_ROOT environment variable: should be defined to point to the rDock installation directory.

• RBT_HOME environment variable: is optional, but can be defined to to point to a user project directory containing rDock input files and customised data files.

• PATH environment variable: $RBT_ROOT/bin should be added to the $PATH environment variable.

• LD_LIBRARY_PATH: $RBT_ROOT/lib should be added to the $LD_LIBRARY_PATH environment variable.

Input file locations. The search path for the majority of input files for rDock is:

• Current working directory

• $RBT_HOME, if defined • The appropriate subdirectory of $RBT_ROOT/data/. For example, the default location for scoring function files is $RBT_ROOT/data/sf/. The exception is that input ligand SD files are always specified as an absolute path. If you wish to customise a scoring function or docking protocol, it is sufficient to copy the relevant file to the current working directory or to $RBT_HOME, and to modify the copied file.

Launching rDock. For small scale experimentation, the rDock executables can be launched directly from the command line. However, serious virtual screening campaigns will likely need access to a compute farm. In common with other docking tools, rDock uses the embarrassingly parallel approach to distributed computing. Large ligand libraries are split into smaller chunks, each of which is docked independently on a single machine. Docking jobs are controlled by a distributed resource manager (DRM) such as Condor or SGE.

## Cavity mapping¶

Virtual screening is very rarely conducted against entire macromolecules. The usual practice is to dock small molecules in a much more confined region of interest. rDock makes a clear distinction between the region the ligand is allowed to explore (known here as the docking site), and the receptor atoms that need to be included in order to calculate the score correctly. The former is controlled by the cavity mapping algorithm, whilst the latter is scoring function dependent as it depends on the distance range of each component term (for example, vdW range >> polar range). For this reason, it is usual practice with rDock to prepare intact receptor files (rather than truncated spheres around the region of interest), and to allow each scoring function term to isolate the relevant receptor atoms within range of the docking site.

rDock provides two methods for defining the docking site:

• Two sphere method

• Reference ligand method

Note All the keywords found in capital letters in following cavity mapping methods explanation (e.g. RADIUS), make reference to the parameters defined in prm rDock configuration file. For more information, go to Cavity mapping subsection in System definition file reference section.

### Two sphere method¶

The two sphere method aims to find cavities that are accessible to a small sphere (of typical atomic or solvent radius) but are inaccessible to a larger sphere. The larger sphere probe will eliminate flat and convex regions of the receptor surface, and also shallow cavities. The regions that remain and are accessible to the small sphere are likely to be the nice well defined cavities of interest for drug design purposes.

1. A grid is placed over the cavity mapping region, encompassing a sphere of radius = RADIUS, center = CENTER. Cavity mapping is restricted to this sphere. All cavities located will be wholly within this sphere. Any cavity that would otherwise protrude beyond the cavity mapping sphere will be truncated at the periphery of the sphere. SLIKA 1

2. Grid points within the volume occupied by the receptor are excluded (coloured red). The radii of the receptor atoms are increased temporarily by VOL_INCR in this step. SLIKA 2

3. Probes of radii LARGE_SPHERE are placed on each remaining unallocated grid point and checked for clashes with receptor excluded volume. To eliminate edge effects, the grid is extended beyond the cavity mapping region by the diameter of the large sphere (for this step only). This allows the large probe to be placed on grid points outside of the cavity mapping region, yet partially protrude into the cavity mapping region. SLIKA 3

4. All grid points within the cavity mapping region that are accessible to the large probe are excluded (coloured green). SLIKA 4

5. Probes of radii SMALL_SPHERE are placed on each remaining grid point and checked for clashes with receptor excluded volume (red) or large probe excluded volume (green) SLIKA 5

6. All grid points that are accessible to the small probe are selected (yellow). SLIKA 6

7. The final selection of cavity grid points is divided into distinct cavities (contiguous regions). In this example only one distinct cavity is found. User-defined filters of MIN_VOLUME and MAX_CAVITIES are applied at this stage to select a subset of cavities if required. Note that the filters will accept or reject intact cavities only. SLIKA 7

### Reference ligand method¶

The reference ligand method provides a much easier option to define a docking volume of a given size around the binding mode of a known ligand, and is particularly appropriate for large scale automated validation experiments.

1. Reference coordinates are read from REF_MOL. A grid is placed over the cavity mapping region, encompassing overlapping spheres of radius = RADIUS, centered on each atom in REF_MOL. Grid points outside of the overlapping spheres are excluded (coloured green). Grid points within the volume occupied by the receptor are excluded (coloured red). The vdW radii of the receptor atoms are increased by VOL_INCR in this step. SLIKA 1

2. Probes of radii SMALL_SPHERE are placed on each remaining grid point and checked for clashes with red or green regions. SLIKA 2

3. All grid points that are accessible to the small probe are selected (yellow). SLIKA 3

4. The final selection of cavity grid points is divided into distinct cavities (contiguous regions). In this example only one distinct cavity is found. User-defined filters of MIN_VOLUME and MAX_CAVITIES are applied at this stage to select a subset of cavities if required. Note that the filters will accept or reject intact cavities only. SLIKA 4

## Scoring function reference¶

### Component Scoring Functions¶

The rDock master scoring function ($$S_{\text{total}}$$) is a weighted sum of intermolecular ($$S_{\text{inter}}$$), ligand intramolecular ($$S_{\text{intra}}$$), site intramolecular ($$S_{\text{site}}$$), and external restraint terms ($$S_{\text{restraint}}$$) ((1)). $$S_{\text{inter}}$$ is the main term of interest as it represents the protein-ligand (or RNA-ligand) interaction score ((2)). $$S_{\text{intra}}$$ represents the relative energy of the ligand conformation ((3)). Similarly, $$S_{\text{site}}$$ represents the relative energy of the flexible regions of the active site ((4)). In the current implementation, the only flexible bonds in the active site are terminal OH and NH3+ bonds. $$S_{\text{restraint}}$$ is a collection of non-physical restraint functions that can be used to bias the docking calculation in several useful ways ((5)).

(1)$S^{\text{total}} = S^{\text{inter}} + S^{\text{intra}} + S^{\text{site}} + S^{\text{restraint}}$
(2)$\begin{split}S^{\text{inter}} = W_{\text{vdW}}^{\text{inter}} \cdot S_{\text{vdW}}^{\text{inter}} + W_{\text{polar}}^{\text{inter}} \cdot S_{\text{polar}}^{\text{inter}} + W_{\text{repul}}^{\text{inter}} \cdot S_{\text{repul}}^{\text{inter}} + W_{\text{arom}}^{\text{inter}} \cdot S_{\text{arom}}^{\text{inter}} +\\ + W_{\text{solv}} \cdot S_{\text{solv}} + W_{\text{rot}} \cdot N_{\text{rot}} + W_{\text{const}}\end{split}$
(3)$S^{\text{intra}} = W_{\text{vdW}}^{\text{intra}} \cdot S_{\text{vdW}}^{\text{intra}} + W_{\text{polar}}^{\text{intra}} \cdot S_{\text{polar}}^{\text{intra}} + W_{\text{repul}}^{\text{intra}} \cdot S_{\text{repul}}^{\text{intra}} + W_{\text{dihedral}}^{\text{intra}} \cdot S_{\text{dihedral}}^{\text{intra}}$
(4)$S^{\text{site}} = W_{\text{vdW}}^{\text{site}} \cdot S_{\text{vdW}}^{\text{site}} + W_{\text{polar}}^{\text{site}} \cdot S_{\text{polar}}^{\text{site}} + W_{\text{repul}}^{\text{site}} \cdot S_{\text{repul}}^{\text{site}} + W_{\text{dihedral}}^{\text{site}} \cdot S_{\text{dihedral}}^{\text{site}}$
(5)$S^{\text{restraint}} = W_{\text{cavity}} \cdot S_{\text{cavity}} + W_{\text{tether}} \cdot S_{\text{tether}} + W_{\text{nmr}} \cdot S_{\text{nmr}} + W_{\text{ph4}} \cdot S_{\text{ph4}}$

$$S_{\text{inter}}$$, $$S_{\text{intra}}$$, and $$S_{\text{site}}$$ are built from a common set of constituent potentials, which are described below. The main changes to the original RiboDock scoring function [RiboDock2004] are:

1. the replacement of the crude steric potentials ($$S_{\text{lipo}}$$ and $$S_{\text{rep}}$$) with a true van der Waals potential, $$S_{\text{vdW}}$$

2. the introduction of two generalised terms for all short range attractive ($$S_{\text{polar}}$$) and repulsive ($$S_{\text{repul}}$$) polar interactions

3. the implementation of a fast weighed solvent accessible surface area (WSAS) solvation term

4. the addition of explicit dihedral potentials

#### van der Waals potential¶

We have replaced the $$S_{\text{lipo}}$$ and $$S_{\text{rep}}$$ empirical potentials used in RiboDock with a true vdW potential similar to that used by GOLD [ref]. Atom types and vdW radii were taken from the Tripos 5.2 force field and are listed in the Appendix section (Table 21). Energy well depths are switchable between the original Tripos 5.2 values and those used by GOLD, which are calculated from the atomic polarisability and ionisation potentials of the atom types involved. Additional atom types were created for carbons with implicit hydrogens, as the Tripos force field uses an all-atom representation. vdW radii for implicit hydrogen types are increased by 0.1 Å for each implicit hydrogen, with well depths unchanged. The functional form is switchable between a softer 4-8 and a harder 6-12 potential. A quadratic potential is used at close range to prevent excessive energy penalties for atomic clashes. The potential is truncated at longer range ($$1.5 \cdot r_{\min}$$, the sum of the vdW radii).

The quadratic potential is used at repulsive energies between $$e_{\text{cutoff}}$$ and $$e_0$$, where $$e_{\text{cutoff}}$$ is defined as a multiple of the energy well depth ($$e_{\text{cutoff}} = \text{ECUT} \cdot e_{\min}$$), and $$e_0$$ is the energy at zero separation, defined as a multiple of $$e_{\text{cutoff}}$$ ($$e_0 = \text{E0} \cdot e_{\text{cutoff}}$$). ECUT can vary between 1 and 120 during the docking search (see Genetic Algorithm subsection in Docking protocol section), whereas E0 is fixed at 1.5.

#### Empirical attractive and repulsive polar potentials¶

We continue to use an empirical Bohm-like potential to score hydrogen-bonding and other short-range polar interactions. The original RiboDock polar terms ($$S_{\text{H-bond}}$$, $$S_{\text{posC-acc}}$$, $$S_{\text{acc-acc}}$$, $$S_{\text{don-don}}$$) are generalised and condensed into two scoring functions, $$S_{\text{polar}}$$ and $$S_{\text{repul}}$$ ((6) and (7), also taking into account (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), and (20)), which deal with attractive and repulsive interactions respectively. Six types of polar interaction centres are considered: hydrogen bond donors (DON), metal ions (M+), positively charged carbons (C+, as found at the centre of guanidinium, amidinium and imidazole groups), hydrogen bond acceptors with pronounced lone pair directionality (ACC_LP), acceptors with in-plane preference but limited lone-pair directionality (ACC_PLANE), and all remaining acceptors (ACC). The ACC_LP type is used for carboxylate oxygens and O sp2 atoms in RNA bases, with ACC_PLANE used for other O sp2 acceptors. This distinction between acceptor types was not made in RiboDock, in which all acceptors were implicitly of type ACC.

(6)$S_{\text{polar}} = \sum_{\text{IC1-IC2}} f_1(|\Delta R_{12}|) \cdot \text{ANG}_{\text{IC1}} \cdot \text{ANG}_{\text{IC2}} \cdot f_2(\text{IC1}) \cdot f_2(\text{IC2}) \cdot f_3(\text{IC1}) \cdot f_3(\text{IC2})$
(7)$S_{\text{repul}} = \sum_{\text{IC1-IC2}} f_1(\Delta R_{12}) \cdot \text{ANG}_{\text{IC1}} \cdot \text{ANG}_{\text{IC2}} \cdot f_2(\text{IC1}) \cdot f_2(\text{IC2}) \cdot f_3(\text{IC1}) \cdot f_3(\text{IC2})$
(8)$\begin{split}f_1(\Delta X) = \begin{cases} 1 & \Delta X \leq \Delta X_{\min}\\ 1 - \frac{\Delta X - \Delta X_{\min}}{\Delta X_{\max} - \Delta X_{\min}} & \Delta X_{\min} < \Delta X \leq \Delta X_{\max}\\ 0 & \Delta X > \Delta X_{\max} \end{cases}\end{split}$
(9)$f_2(i) = sgn(i)(1 + 0.5 |c_i|)$
(10)$\begin{split}sgn(i) = \begin{cases} -1 & \text{ACC}, \text{ACC}\_\text{LP}, \text{ACC}\_\text{PLANE}\\ +0.5 & \text{C+}\\ +1.0 & \text{DON}, \text{M+} \end{cases}\end{split}$
(11)$c_i = \text{formal charge on primary atom of interaction centre $$i$$}$
(12)$\begin{split}f_3(\Delta X) = \begin{cases} \sqrt{\frac{N_i}{25}} & \text{macromolecular interaction centres}\\ 1 & \text{ligand interaction centres} \end{cases}\end{split}$
(13)$\begin{split}N_i = \text{number of non-hydrogen macromolecule atoms within}\\ \text{5 Å radius of primary atom of interaction centre $$i$$}\end{split}$

Individual interaction scores are the product of simple scaling functions for geometric variables, formal charges and local neighbour density. The scaling functions themselves, and the formal charge assignment method, are retained from RiboDock. Metals are assigned a uniform formal charge of +1. C+ is considered to be a weak donor in this context and scores are scaled by 50 % relative to conventional donors by the assignment of $$sgn(i) = 0.5$$ in (9). Pseudo-formal charges are no longer assigned to selected RNA base atoms. The geometric functions minimally include an interaction distance term, with the majority also including angular terms dependent on the type of the interaction centres. Geometric parameters and the angular functions are summarised in Appendix section (Table 22 and Table 23, respectively).

The most notable improvements to RiboDock are that attractive (hydrogen bond and metal) interactions with ACC_LP and ACC_PLANE acceptors include terms for $$\phi$$ and $$\theta$$ (as defined in [ref 3]) to enforce the relevant lone pair directionality. These replace the $$\alpha_{\text{ACC}}$$ dependence, which is retained for the ACC acceptor type. No distinction between acceptor types is made for attractive interactions with C+ carbons, or for repulsive interactions between acceptors. In these circumstances all acceptors are treated as type ACC. Such C+-ACC interactions, which in RiboDock were described by only a distance function, ($$S_{\text{posC-acc}}$$) now include angular functions around the carbon and acceptor groups. Repulsive interactions between donors, and between acceptors, also have an angular dependence. This allows a stronger weight, and a longer distance range, to be used to penalise disallowed head-to-head interactions without forbidding allowable contacts. One of the issues in RiboDock was that it was not possible to include neutral acceptors in the acceptor-acceptor repulsion term with a simple distance function.

#### Solvation potential¶

The desolvation potential in rDock combines a weighted solvent accessible surface area approach [WSAS [ref4]] with a rapid probabilistic approximation to the calculation of solvent accessible surface areas [ref5] based on pairwise interatomic distances and radii ((14), taking into account (15), (16), (17), (18), (19), and (20)).

(14)$S_{\text{solv}} = (\Delta G_{\text{WSAS}}^{\text{site,bound}} - \Delta G_{\text{WSAS}}^{\text{site$$_0$$,unbound}}) + (\Delta G_{\text{WSAS}}^{\text{ligand,bound}} - \Delta G_{\text{WSAS}}^{\text{ligand$$_0$$,unbound}})$
(15)$r_{\text{s}} = 0.6 \text{Å}$
(16)$\begin{split}p_{ij} = \begin{cases} 0.8875 & \quad \text{1-2 intramolecular connections}\\ 0.3516 & \quad \text{1-3 intramolecular connections}\\ 0.3156 & \quad \text{1-4 intramolecular connections and above}\\ 0.3156 & \quad \text{intermolecular interactions} \end{cases}\end{split}$
(17)$S_i = 4 \pi (r_i + r_s)^2$
(18)$b_{ij} = \pi (r_i + r_s) (r_j + r_i + 2 r_s - d) \Big(1 - \frac{r_j - r_i}{d}\Big)$
(19)$A_i = S_i \prod_j 1 - \frac{p_i p_{ij} b_{ij}}{S_i}$
(20)$\Delta G_{\text{WSAS}} = \sum_{i=1}^{n_i} w_i A_i$

The calculation is fast enough therefore to be used in docking. We have redefined the solvation atom types compared to the original work [ref4] and recalibrated the weights against the same training set of experimental solvation free energies in water (395 molecules). The total number of atom types (50, including 6 specifically for ionic groups and metals) is slightly lower than in original work (54). Our atom types reflect the fact that rDock uses implicit non-polar hydrogens. The majority of types are a combination of hybridisation state and the number of implicit or explicit hydrogens. All solvation parameters are listed in Appendix section (Table 24).

$$S_{\text{solv}}$$ is calculated as the change in solvation energy of the ligand and the docking site upon binding of the ligand. The reference energies are taken from the initial conformations of the ligand and site (as read from file) and not from the current pose under evaluation. This is done to take into account any changes to intramolecular solvation energy. Strictly speaking the intramolecular components should be reported separately under $$S_{\text{intra}}$$ and $$S_{\text{site}}$$ but this is not done for reasons of computational efficiency.

#### Dihedral potential¶

Dihedral energies are calculated using Tripos 5.2 dihedral parameters for all ligand and site rotatable bonds. Corrections are made to account for the missing contributions from the implicit non-polar hydrogens.

### Intermolecular scoring functions under evaluation¶

#### Training sets¶

We constructed a combined set of protein-ligand and RNA-ligand complexes for training of rDock. Molecular data files for the protein-ligand complexes were extracted from the downloaded CCDC/Astex cleanlist[ref6] and used without substantive modification. The only change was to convert ligand MOL2 files to MDL SD format using Corina [ref], leaving the coordinates and protonation states intact.

Protein MOL2 files were read directly. The ten RNA-ligand NMR structures from the RiboDock validation set were supplemented with five RNA-ligand crystal structures (1f1t, 1f27, 1j7t, 1lc4, 1mwl) prepared in a similar way. All 15 RNA-ligand structures have measured binding affinities.

58 complexes (43 protein-ligand and 15 RNA-ligand) were selected for the initial fitting of component scoring function weights. Protein-ligand structures were chosen (of any X-ray resolution) that had readily available experimental binding affinities [ref 7].102 complexes were used for the main validation of native docking accuracy for different scoring function designs, consisting of 87 of the 92 entries in the high resolution (R<2 Å) clean-list (covalently bound ligands removed – 1aec, 1b59, 1tpp, 1vgc, 4est), and the 15 RNA-ligand complexes.

#### Scoring functions design¶

Component weights ($$W$$) for each term in the intermolecular scoring function ($$S_{\text{inter}}$$) were obtained by least squares regression of the component scores to $$\Delta G_{\text{bind}}$$ values for the binding affinity training set described above (58 entries). Each ligand was subjected first to simplex minimisation in the docking site, starting from the crystallographic pose, to relieve any minor non-bonded clashes with the site. The scoring function used for minimisation was initialised with reasonable manually assigned weights. If the fitted weights deviated significantly from the initial weights the procedure was repeated until convergence. Certain weights ($$W_{\text{repul}}$$, $$W_{\text{rot}}$$, $$W_{\text{const}}$$) were constrained to have positive values to avoid non-physical, artefactual models. Note that the presence of $$W_{\text{rot}}$$ and $$W_{\text{const}}$$ in $$S_{\text{inter}}$$ improves the quality of the fit to the binding affinities but does not impact on native ligand docking accuracy.

Ten intermolecular scoring functions were derived with various combinations of terms (Table 4). SF0 is a baseline scoring function that has the van der Waals potential only. SF1 adds a simplified polar potential, without f2 (formal charge) and f3 (neighbour density) scaling functions, and with a single acceptor type (ACC) that lacks lone-pair directionality. SF2 has the full polar potential (f2 and f3 scaling functions, ACC, ACC_LP and ACC_PLANE acceptor types) and adds the repulsive polar potential. SF3 has the same functional form as SF2 but with empirical weights in regular use at RiboTargets. SF4 replaces the repulsive polar potential with the WSAS desolvation potential described above. SF5 has the same functional form as SF4 but with empirical weights in regular use at RiboTargets. SF6 combines the repulsive polar and desolvation potentials. SF7 has the same functional form as SF2 and SF3 but with weights for $$W_{\text{vdW}}$$ and $$W_{\text{polar}}$$ taken from SF5. SF8 and SF9 add the crude aromatic term from RiboDock [RiboDock2004] to SF3 and SF5 respectively. The $$S_{\text{intra}}$$ functional form and weights were held constant, and equivalent to SF3, to avoid any differences in ligand conformational energies affecting the docking results. As the $$S_{\text{site}}$$ scores are calculated simultaneously with $$S_{\text{inter}}$$ (for computational reasons) the $$S_{\text{site}}$$ functional form and weights vary in line with $$S_{\text{inter}}$$. There is surprisingly little variation in correlation coefficient (R) and root mean square error (RMSE) in predicted binding energy over the ten scoring functions (Table 4). The best results are obtained with SF4 (R = 0.67, RMSE = 9.6 kJ/mol).

Table 4 Intermolecular scoring function weights under evaluation (a = constrained to be > zero; b = fixed values; c = correlation coefficient (R), and root mean squared error (RMSE) between $$S_{\text{inter}}$$ and $$\Delta G_{\text{bind}}$$, for minimised experimental ligand poses, over binding affinity validation set (58 entries)).

SF

$$W_{\text{vdW}}$$

$$W_{\text{polar}}$$

$$W_{\text{solv}}$$

$$W_{\text{repul}}$$a

$$W_{\text{arom}}$$

$$W_{\text{rot}}$$a

$$W_{\text{const}}$$a

Rc

RMSEc

0

1.4

-

-

-

-

0

0

0.62

10.9

1

1.126

2.36

-

-

-

0.217

0

0.64

10.2

2

1.192

2.087

-

2.984

-

0

0

0.63

10.4

3

1.000b

3.400b

-

5.000b

-

0

0

0.59

10.9

4

1.317

3.56

0.449

-

-

0

4.

0.67

9.6

5

1.500b

5.000b

0.500b

-

-

0.568

4.782

0.62

10.7

6

1.314

4.447

0.500b

5.000b

-

0

0

0.62

10.4

7

1.500b

5.000b

-

5.000b

-

0.986

12.046

0.55

12.9

8

1.000b

3.400b

-

5.000b

-1.6b

0

0

0.53

11.8

9

1.500b

5.000b

0.500b

-

-1.6b

0.647

5.056

0.58

11.5

#### Scoring functions validation¶

The ability of the ten intermolecular scoring functions (SF0 to SF9) to reproduce known ligand binding modes was determined on the combined test set of 102 protein-ligand and RNA-ligand complexes. The intra-ligand scoring function ($$S_{\text{intra}}$$) was kept constant, with component weights equivalent to SF3, and a dihedral weight of 0.5. Terminal OH and NH3 groups on the receptor in the vicinity of the docking site were fully flexible during docking. Ligand pose populations of size $$N_{\text{pop}} = 300$$ were collected for each complex and intermolecular scoring function combination. The population size was increased to $$N_{\text{pop}} = 1000$$ for two of the most promising scoring functions (SF3 and SF5).

Protein-ligand docking accuracy is remarkably insensitive to scoring function changes. Almost half of the ligand binding modes can be reproduced with a vdW potential only (SF0). The addition of a simplified polar potential (SF1) increases the accuracy to over 70 % of protein-ligand test cases predicted to within 2 Å RMSD. The success rate increases further to 78 % with SF3, which has the full attractive and repulsive polar potentials, and empirically adjusted weights relative to SF2. Subsequent changes to the component terms and weights, including the addition of the desolvation potential, have little or no impact on the protein-ligand RMSD metric.

The nucleic acid set shows a much greater sensitivity to scoring function changes. This can in part be explained by the smaller sample size that amplifies the percentage changes in the RMSD metric, but nevertheless the trends are clear. There is a gradual increase in docking accuracy from SF0 (37 %) to SF3 (52 %), but absolute performance is much lower than for the protein-ligand test set. This level of docking accuracy for nucleic acid-ligand complexes is broadly consistent with the original RiboDock scoring function, despite the fact that the original steric term (LIPO) has been replaced by a true vdW potential. The introduction of the desolvation potential in place of the empirical repulsive polar potential (in SF4 and SF5) results in a substantial improvement in accuracy, to around 70 % of test cases within 2 Å RMSD. Subsequent changes (SF6 to SF9) degrade the accuracy. The lower performance of SF7, which has the higher weights for the VDW and POLAR terms taken from SF5 but lacks the desolvation potential, demonstrates that it is the desolvation term itself that is having the beneficial effect, and not merely the reweighting of the other terms. The inclusion of the geometric aromatic term in SF8 and SF9 has a detrimental impact on the performance of SF3 and SF5 respectively.

Overall, SF5 achieves optimum performance across proteins and nucleic acids (76.7 % within 2 Å RMSD). SF3 (no desolvation potential) and SF5 (with desolvation potential) were selected as the best intermolecular scoring functions. Finally, these two scoring functions, SF3 and SF5, were the ones implemented in rDock with the names of dock.prm and dock_solv.prm, respectively.

Note In Virtual Screening campaigns, or in experiments where score of different ligands is compared, the best scoring poses for each molecule (as defined by the lowest $$S_{\text{total}}$$ within the sample) are sorted and ranked by $$S_{\text{inter}}$$. In other words, the contributions to $$S_{\text{total}}$$ from $$S_{\text{intra}}$$, $$S_{\text{site}}$$ and $$S_{\text{restraint}}$$ are ignored when comparing poses between different ligands against the same target. The rationale for this is that, in particular, the ligand intramolecular scores are not on an absolute scale and can differ markedly between different ligands.

### Code implementation¶

Scoring functions for docking are constructed at run-time (by RbtSFFactory class) from scoring function definition files (rDock .prm format). The default location for scoring function definition files is $RBT_ROOT/data/sf/. The total score is an aggregate of intermolecular ligand-receptor and ligand-solvent interactions (branch SCORE.INTER), intra-ligand interactions (branch SCORE.INTRA), intra-receptor, intra-solvent and receptor-solvent interactions (branch SCORE.SYSTEM), and external restraint penalties (branch SCORE.RESTR). The SCORE.INTER, SCORE.INTRA and SCORE.SYSTEM branches consist of weighted sums of interaction terms as shown below. Note that not all terms appear in all branches. See the rDock draft paper for more details on the implementation of these terms. Table 5 Scoring function terms and C++ implementation classes. Term Description INTER INTRA SYSTEM VDW van der Waals RbtVdWIdxSF RbtVdwIntraSF RbtVdwIdxSF VDW van der Waals (grid based) RbtVdwGridSF N/A N/A POLAR Attractive polar RbtPolarIdxSF RbtPolarIntraSF RbtPolarIdxSF REPUL Repulsive polar RbtPolarIdxSF RbtPolarIntraSF RbtPolarIdxSF SOLV Desolvation RbtSAIdxSF RbtSAIdxSF RbtSAIdxSF CONST Translation/rotational binding entropy penalty RbtConstSF N/A RbtConstSF ROT Torsional binding entropy penalty RbtRotSF N/A N/A Two intermolecular scoring functions (SCORE.INTER branch) have been validated. These are known informally as the standard scoring function and the desolvation scoring function (referred to as SF3 and SF5 respectively in the rDock draft paper). The standard intermolecular scoring function consists of VDW, POLAR and REPUL terms. In the desolvation scoring function, the REPUL term is replaced by a more finely parameterised desolvation potential (SOLV term) based on a Weighted Solvent-Accessible Surface Area (WSAS) model. The ligand intramolecular scoring function (SCORE.INTRA branch) remains constant in both cases, and has the same terms and weights as the standard intermolecular scoring function. Table 6 Scoring function data files. File Description RbtInterIdxSF.prm Intermolecular scoring function definition (standard scoring function, SF3) RbtInterGridSF.prm As above, but vdW term uses a precalculated grid RbtSolvIdxSF.prm Intermolecular scoring function definition (desolvation scoring function, SF5) calcgrid_vdw1.prm vdW term only (ECUT = 1), for calculating vdW grid (used by rbcalcgrid) calcgrid_vdw5.prm vdW term only (ECUT = 5), for calculating vdW grid (used by rbcalcgrid) Tripos52_vdw.prm vdW term parameter file Tripos52_dihedrals.prm Dihedral term parameter file solvation_asp.prm Desolvation term parameter file Note External restraint penalty terms are defined by the user in the system definition .prm file. Originally, rDock did not support flexible receptor dihedrals or explicit structural waters, and the overall scoring function consisted of just the SCORE.INTER and SCORE.INTRA branches. At that time, the intermolecular scoring function definition file (e.g. RbtInterIdxSF.prm) represented precisely the SCORE.INTER terms, and the intramolecular definition file (RbtIntraSF.prm) represented precisely the SCORE.INTRA terms. With the introduction of receptor flexibility and explicit structural waters (and hence the need for the SCORE.SYSTEM branch), the situation is slightly more complex. For implementation reasons, many of the terms reported under SCORE.SYSTEM (with the exception of the dihedral term) are calculated simultaneously with the terms reported under SCORE.INTER, and hence their parameterisation is defined implicitly in the intermolecular scoring function definition file. In contrast, the ligand intramolecular scoring function terms can be controlled independently. ## Docking protocol¶ ### Protocol summary¶ #### Pose generation¶ rDock uses a combination of stochastic and deterministic search techniques to generate low energy ligand poses. The standard docking protocol to generate a single ligand pose uses 3 stages of Genetic Algorithm search (GA1, GA2, GA3), followed by low temperature Monte Carlo (MC) and Simplex minimisation (MIN) stages. Several scoring function parameters are varied between the stages to promote efficient sampling. The ECUT parameter of the $$S_{\text{inter}}$$ vdW potential (defining the hardness of the intermolecular close range potential) is increased from 1 in the first GA stage (GA1) to a maximum of 120 in the MC and MIN stages, with intermediate values of 5 in GA2 and 25 in GA3. The functional form of the $$S_{\text{inter}}$$ vdW potential is switched from a 4-8 potential in GA1 and GA2 to a 6-12 potential in GA3, MC and MIN. In a similar fashion, the overall weight of the $$S_{\text{intra}}$$ dihedral potential is ramped up from an initial value of 0.1 in GA1 to a final value of 0.5 in the MC and MIN stages, with intermediate values of 0.2 in GA2 and 0.3 in GA3. In contrast, the $$S_{\text{intra}}$$ vdW parameters (as used for the ligand intramolecular potential) remain fixed at the final, hard values throughout the calculation (ECUT = 120, 6-12 potential). Overall, we found this combination of parameter changes allows for efficient sampling of the very poor starting poses, whilst minimising the likelihood that poor ligand internal conformations are artificially favoured and trapped early in the search, and ensures that physically realistic potentials are used for final optimisation and analysis. #### Genetic Algorithm¶ The GA chromosome consists of the ligand centre of mass (com), the ligand orientation, as represented by the quaternion (q) required to rotate the ligand principal axes from the Cartesian reference axes, the ligand rotatable dihedral angles, and the receptor rotatable dihedral angles. The ligand centre of mass and orientation descriptors, although represented by multiple floating point values (com.x, com.y, com.z, and q.s, q.x, q.y, q.z respectively), are operated on as intact entities by the GA mutation and crossover operators. For so-called free docking, in which no external restraints other than the cavity penalty are imposed, the initial population is generated such that the ligand centre of mass is constrained to lie on a randomly selected grid point within the defined docking volume, and the ligand orientation and all dihedral angles are randomised completely. Mutations to the ligand centre of mass are by a random distance along a randomly oriented unit vector. Mutations to the ligand orientation are performed by rotating the ligand principal axes by a random angle around a randomly oriented unit vector. Mutations to the ligand and receptor dihedral angles are by a random angle. All mutation distances and angles are randomly selected from rectangular distributions of defined width. A generation is considered to have passed when the number of new individuals created is equal to the population size. Instead of having a fixed number of generations, the GA is allowed to continue until the population converges. The population is considered converged when the score of the best scoring pose fails to improve by more than 0.1 over the last three generations. This allows early termination of poorly performing runs for which the initial population is not able to generate a good solution. During initial testing the impact of a wide variety of GA parameters (Table 7) were explored on a small, representative set of protein-ligand complexes (3ptb, 1rbp, 1stp, 3dfr). We measured the frequency that the algorithm was able to find the experimental conformation, and the average run time. Optimum results were obtained with a steady state GA, roulette wheel selection, a single population of size ($$100 \times \text{(number of rotatable bonds)}$$), a crossover:mutation ratio of 40:60, and mutation distribution widths of ligand translation 2 Å, ligand rotation of 30 degrees and dihedral angle of 30 degrees. These parameters have been found to be generally robust across a wide variety of systems. Table 7 Summary of GA parameter space explored, and final values Parameter Values Explored Final Values Number of populations 1, 2, 3, 4, 5 1 Selection operator Roulette wheel, Rank Roulette wheel Mutation Rectangular Cauchy Rectangular GA Generational, Steady state Steady state Elitism Yes, No No No of individuals modified in each generation All values from 1 to population size 0.5 * population size Population size 50, 75, 100, 125, 150, 200, 400, 800 * number of rotatable bonds 100 * number of rotatable bonds Probability of choosing Crossover vs. Mutation 0.0, 0.05, 0.1 … 0.9, 0.95, 1.0 0.4 Torsion step 3, 12, 21, 30 degrees 30 degrees Rotational step 3, 12, 21, 30 degrees 30 degrees Translation step 0.1, 0.8, 1.4, 2.0 Å 2.0 Å #### Monte Carlo¶ The method and parameters for low temperature Monte Carlo are similar to those described for phase 4 of the RiboDock simulated annealing search protocol. The overall number of trials is scaled according to the number of rotatable bonds in the ligand, from a minimum of 500 ($$N_{\text{rot}} = 0$$) to a maximum of 2000 ($$N_{\text{rot}} = 15$$). Maximum step sizes are: translation 0.1 Å, ligand rotation of 10 degrees and dihedral angle of 10 degrees. Step sizes are halved if the Metropolis acceptance rate falls below 0.25. #### Simplex¶ The Nelder-Mead’s Simplex minimisation routine operates on the same chromosome representation as the GA, with the exception that the composite descriptors (centre of mass and orientation) are decomposed into their constituent floating point values. ### Code implementation¶ Docking protocols are constructed at run-time (by RbtTransformFactory class) from docking protocol definition files (rDock .prm format). The default location for docking protocol files is $RBT_ROOT/data/scripts/. The docking protocol definition file defines the sequence of search algorithms that constitute a single docking run for a single ligand record. Each search algorithm component operates either on a single chromosome representing the system degrees of freedom, or on a population of such chromosomes. The chromosome is constructed (by RbtChromFactory class) as an aggregate of individual chromosome elements for the receptor, ligand and explicit solvent degrees of freedom, as defined by the flexibility parameters in the system definition file.

Table 8 Chromosome elements

Element

Defined by

Class

Length

Position

Centre of mass

RbtChromPositionElement

3

Orientation

Euler angles for principal axes

RbtChromPositionElement

3

Dihedral

Dihedral angle for rotatable bond

RbtChromDihedralElement

1 per bond

Occupancy

Explicit water occupancy state

RbtChromOccupancylElement

1 per water

### Standard rDock docking protocol (dock.prm)¶

As stated above in this section, the standard rDock docking protocol consists of three phases of a Genetic Algorithm search, followed by low-temperature Monte Carlo and Simplex minimisation.

Table 9 Search algorithm components and C++ implementation classes

Component

Class

Operates on

Randomise population

RbtRandPopTransform

Chromosome population

Genetic algorithm search

RbtGATransform

Chromosome population

Monte Carlo simulated annealing

RbtSimAnnTransform

Single chromosome

Simplex minimisation

RbtSimplexTransform

Single chromosome

Null operation

RbtNullTransform

N/A

Table 10 Docking protocol data files

File

Description

score.prm

Calculates score only for initial conformation (standard scoring function)

scole_solv.prm

As above, but uses desolvation scoring function

minimise.prm

Simplex minimisation of initial conformation (standard scoring function)

minimise_solv.prm

As above, but uses desolvation scoring function

dock.prm

Full docking search (standard scoring function)

dock_solv.prm

As above, but uses desolvation scoring function

dock_grid.prm

Full docking search (standard scoring function, grid-based vdW term)

dock_solv_grid.prm

Full docking search (desolvation scoring function, grid-based vdW term)

By way of example, the dock.prm script is documented in detail. The other scripts are very similar.

SECTION SCORE
INTER RbtInterIdxSF.prm
INTRA RbtIntraSF.prm
SYSTEM RbtTargetSF.prm
END_SECTION


Scoring Function The scoring function definition is referenced within the docking protocol definition file itself, in the SCORE section. This section contains entries for the INTER, INTRA and SYSTEM scoring function definition files.

SECTION SETSLOPE_1
TRANSFORM RbtNullTransform
# Dock with a high penalty for leaving the cavity
WEIGHT@SCORE.RESTR.CAVITY 5.0
# Gradually ramp up dihedral weight from 0.1-->0.5
WEIGHT@SCORE.INTRA.DIHEDRAL 0.1
# Gradually ramp up energy cut off for switching to quadratic
ECUT@SCORE.INTER.VDW 1.0
# Start docking with a 4-8 vdW potential
USE 4_8@SCORE.INTER.VDW TRUE
# Broader angular dependence
DA1MAX@SCORE.INTER.POLAR 180.0
# Broader angular dependence
DA2MAX@SCORE.INTER.POLAR 180.0
# Broader distance range
DR12MAX@SCORE.INTER.POLAR 1.5
END_SECTION


Genetic Algorithm All sections that contain the TRANSFORM parameter are interpreted as a search algorithm component. The value of the TRANSFORM parameter is the C++ implementation class name for that transform. An RbtNullTransform can be used to send messages to the scoring function to modify key scoring function parameters in order to increase search efficiency. All parameter names that contain the @ symbol are interpreted as scoring function messages, where the string before the @ is the scoring function parameter name, the string after the @ is the scoring function term, and the parameter value is the new value for the scoring function parameter. Messages are sent blind, with no success feedback, as the docking protocol has no knowledge of the composition of the scoring function terms.

Here, we start the docking with a soft 4-8 vdW potential, a reduced dihedral potential, and extended polar ranges (distances and angles) for the intermolecular polar potential. These changes are all designed to aid sampling efficiency by not overpenalising bad contacts in the initial, randomised population, and by encouraging the formation of intermolecular hydrogen bonds.

SECTION RANDOM_POP
TRANSFORM RbtRandPopTransform
POP_SIZE 50
SCALE_CHROM_LENGTH TRUE
END_SECTION


Creates an initial, randomised chromosome population. If SCALE_CHROM_LENGTH is false, the population is of fixed size, defined by POP_SIZE. If SCALE_CHROM_LENGTH is true, the population is proportional to the overall chromosome length, defined by POP_SIZE multiplied by the chromosome length.

SECTION GA_SLOPE1
TRANSFORM RbtGATransform
PCROSSOVER 0.4 # Prob. of crossover
XOVERMUT TRUE # Cauchy mutation after each crossover
CMUTATE FALSE # True = Cauchy ; False = Rectang. for regular mutations
STEP_SIZE 1.0 # Max relative mutation
END_SECTION


First round of GA.

SECTION SETSLOPE_3
TRANSFORM RbtNullTransform
WEIGHT@SCORE.INTRA.DIHEDRAL 0.2
ECUT@SCORE.INTER.VDW 5.0
DA1MAX@SCORE.INTER.POLAR 140.0
DA2MAX@SCORE.INTER.POLAR 140.0
DR12MAX@SCORE.INTER.POLAR 1.2
END_SECTION


Increases the ligand dihedral weight, increases the short-range intermolecular vdW hardness (ECUT), and decreases the range of the intermolecular polar distances and angles.

SECTION GA_SLOPE3
TRANSFORM RbtGATransform
PCROSSOVER 0.4 # Prob. of crossover
XOVERMUT TRUE # Cauchy mutation after each crossover
CMUTATE FALSE # True = Cauchy ; False = Rectang. for regular mutations
STEP_SIZE 1.0 # Max relative mutation
END_SECTION


Second round of GA with revised scoring function parameters.

SECTION SETSLOPE_5
TRANSFORM RbtNullTransform
WEIGHT@SCORE.INTRA.DIHEDRAL 0.3
ECUT@SCORE.INTER.VDW 25.0
# Now switch to a convential 6-12 for final GA, MC, minimisation
USE 4_8@SCORE.INTER.VDW FALSE
DA1MAX@SCORE.INTER.POLAR 120.0
DA2MAX@SCORE.INTER.POLAR 120.0
DR12MAX@SCORE.INTER.POLAR 0.9
END_SECTION


Further increases the ligand dihedral weight, further increases the short-range intermolecular vdW hardness (ECUT), and further decreases the range of the intermolecular polar distances and angles. Also switches from softer 4-8 vdW potential to a harder 6-12 potential for final round of GA, MC and minimisation.

SECTION GA_SLOPE5
TRANSFORM RbtGATransform
PCROSSOVER 0.4 # Prob. of crossover
XOVERMUT TRUE # Cauchy mutation after each crossover
CMUTATE FALSE # True = Cauchy ; False = Rectang. for regular mutations
STEP_SIZE 1.0 # Max relative mutation
END_SECTION


Final round of GA with revised scoring function parameters.

SECTION SETSLOPE_10
TRANSFORM RbtNullTransform
WEIGHT@SCORE.INTRA.DIHEDRAL 0.5 # Final dihedral weight matches SF file
ECUT@SCORE.INTER.VDW 120.0 # Final ECUT matches SF file
DA1MAX@SCORE.INTER.POLAR 80.0
DA2MAX@SCORE.INTER.POLAR 100.0
DR12MAX@SCORE.INTER.POLAR 0.6
END_SECTION


Resets all the modified scoring function parameters to their final values, corresponding to the values in the scoring function definition files. It is important that the final scoring function optimised by the docking search can be compared directly with the score-only and minimisation-only protocols, in which the scoring function parameters are not modified.

SECTION MC_10K
TRANSFORM RbtSimAnnTransform
START_T 10.0
FINAL_T 10.0
NUM_BLOCKS 5
STEP_SIZE 0.1
MIN_ACC_RATE 0.25
PARTITION_DIST 8.0
PARTITION_FREQ 50
HISTORY_FREQ 0
END_SECTION


Monte Carlo Low temperature Monte Carlo sampling, starting from fittest chromosome from final round of GA.

SECTION SIMPLEX
TRANSFORM RbtSimplexTransform
MAX_CALLS 200
NCYCLES 20
STOPPING_STEP_LENGTH 10e-4
PARTITION_DIST 8.0
STEP_SIZE 1.0
CONVERGENCE 0.001
END_SECTION


Minimisation Simplex minimisation, starting from fittest chromosome from low temperature Monte Carlo sampling.

SECTION FINAL
TRANSFORM RbtNullTransform
WEIGHT@SCORE.RESTR.CAVITY 1.0 # revert to standard cavity penalty
END_SECTION


Finally, we reset the cavity restraint penalty to 1. The penalty has been held at a value of 5 throughout the search, to strongly discourage the ligand from leaving the docking site.

## System definition file reference¶

Although known previously as the receptor .prm file, the system definition file has evolved to contain much more than the receptor information. The system definition file is used to define:

### Receptor definition¶

The receptor can be loaded from a single MOL2 file, or from a combination of Charmm PSF and CRD files. In the former case the MOL2 file provides the topology and reference coordinates simultaneously, whereas in the latter case the topology is loaded from the PSF file and the reference coordinates from the CRD file. For historical compatibility reasons, receptor definition parameters are all defined in the top-level namespace and should not be placed between SECTION / END_SECTION pairs.

Caution If MOL2 and PSF/CRD parameters are defined together, the MOL2 parameters take precedence and are used to load the receptor model.

Table 11 Receptor definition parameters

Parameter

Description

Type

Default

Range of values

Parameters specific to loading receptor in MOL2 file format

RECEPTOR_FILE

Name of receptor MOL2 file

Filename string

No default value

Valid MOL2 filename

Parameters specific to loading receptor in Charmm PSF/CRD file format

RECEPTOR_TOPOL_FILE

Name of receptor Charmm PSF file

Filename string

No default value

Valid Charmm PSF filename

RECEPTOR_COORD_FILE

Name of receptor Charmm CRD file

Filename string

No default value

Valid Charmm CRD filename

RECEPTOR_MASSES_FILE

Name of rDock-annotated Charmm masses file

Filename string

No default value

masses.rtf, top_all2_prot_na.inp

General receptor parameters, applicable to either file format

RECEPTOR_SEGMENT_NAME

List of molecular segment names to read from either MOL2 or PSF/CRD file. If this parameter is defined, then any segment/chains not listed are not loaded. This provides a convenient way to remove cofactors, counterions and solvent without modifying the original file.

Comma separated list of segment name strings (without any spaces)

Empty (i.e. all segments read from file)

Comma separated list of segment name strings

RECEPTOR_FLEX

Defines terminal OH and NH3+ groups withing this distance of docking volume as flexible.

float (Angstroms)

Undefined (rigid receptor)

>0.0 (3.0 is a reasonable value)

Advanced parameters (should not need to be changed by the user)

RECEPTOR_ALL_H

Disable the removal of explicit non-polar hydrogens from the receptor model. Not recommended

boolean

FALSE

TRUE or FALSE

DIHEDRAL_STEP

Maximum mutation step size for receptor dihedral degrees of freedom.

float (degrees)

30.0

>0.0

### Ligand definition¶

Ligand definition parameters need only be defined if you wish to introduce tethering of some or all of the ligand degrees of freedom. If you are running conventional free docking then this section is not required. All ligand definition parameters should be defined in SECTION LIGAND. Note that the ligand input SD file continues to be specified directly on the rbdock command-line and not in the system definition file.

Table 12 Ligand definition parameters

Parameter

Description

Type

Default

Range of values

Main user parameters

TRANS_MODE

Sampling mode for ligand translational degrees of freedom

enumerated string literal

FREE

FIXED, TETHERED, FREE

ROT_MODE

Sampling mode for ligand whole-body rotational degrees of freedom

enumerated string literal

FREE

FIXED, TETHERED, FREE

DIHEDRAL_MODE

Sampling mode for ligand internal dihedral degrees of freedom

enumerated string literal

FREE

FIXED, TETHERED, FREE

MAX_TRANS

(for TRANS_MODE = TETHERED only) Maximum deviation allowed from reference centre of mass

float (Angstroms)

1.0

>0.0

MAX_ROT

(for ROT_MODE = TETHERED only) Maximum deviation allowed from orientation for reference principle axes

float (degrees)

30.0

>0.0–180.0

MAX_DIHEDRAL

(for DIHEDRAL_MODE = TETHERED only) Maximum deviation allowed from reference dihedral angles for any rotatable bond

float (degrees)

30.0

>0.0–180.0

Advanced parameters (should not need to be changed by the user)

TRANS_STEP

Maximum mutation step size for ligand translational degrees of freedom

float (Angstroms)

2.0

>0.0

ROT_STEP

Maximum mutation step size for ligand whole-body rotational degrees of freedom

float (degrees)

30.0

>0.0

DIHEDRAL_STEP

Maxium mutation step size for ligand internal dihedral degrees of freedom

float (degrees)

30.0

>0.0

### Solvent definition¶

Solvent definition parameters need only be defined if you wish to introduce explicit structural waters into the docking calculation, otherwise this section is not required. All solvent definition parameters should be defined in SECTION SOLVENT.

Table 13 Solvent definition parameters

Parameter

Description

Type

Default

Range of values

Main user parameters

FILE

Name of explicit solvent PDB file

File name string

No default value (mandatory parameter)

Valid PDB filename

TRANS_MODE

Sampling mode for solvent translational degrees of freedom. If defined here, the value overrides the per-solvent translational sampling modes defined in the solvent PDB file

enumerated string literal

FREE

FIXED, TETHERED, FREE

ROT_MODE

Sampling mode for solvent whole-body rotational degrees of freedom. If defined here, the value overrides the per-solvent rotational sampling modes defined in the solvent PDB file

enumerated string literal

FREE

FIXED, TETHERED, FREE

MAX_TRANS

(for TRANS_MODE = TETHERED waters only) Maximum deviation allowed from reference water oxygen positions. The same value is applied to all waters with TRANS_MODE = TETHERED; it is no possible currently to define per-solvent MAX_TRANS values

float (Angstroms)

1.0

>0.0

MAX_ROT

(for ROT_MODE = TETHERED waters only) Maximum deviation allowed from orientation of reference principal axes. The same value is applied to all waters with ROT_MODE = TETHERED; it is no possible currently to define per-solvent MAX_ROT values

float (degrees)

30.0

>0.0–180.0

OCCUPANCY

Controls occupancy state sampling for all explicit solvent. If defined here, the values overrides the per-solvent occupancy states defined in the solvent PDB file

float

1.0

0.0–1.0

Advanced parameters (should not need to be changed by the user)

TRANS_STEP

Maximum mutation step size for solvent translational degrees of freedom

float (Angstroms)

2.0

>0.0

ROT_STEP

Maximum mutation step size for solvent wholebody rotational degrees of freedom

float (degrees)

30.0

>0.0

OCCUPANCY_STEP

Maximum mutation step size for solvent occupancy state degrees of freedom

float (degrees)

1.0

0.0–1.0

Solvent occupancy state sampling OCCUPANCY = 0 permanently disables all solvent; OCCUPANCY = 1.0 permanently enables all solvent; OCCUPANCY between 0 and 1 activates variable occupancy state sampling, where the value represents the initial probability that the solvent molecule is enabled. For example, OCCUPANCY = 0.5 means that the solvent is enabled in 50 % of the initial GA population. However, the probability that the solvent is actually enabled in the final docking solution will depend on the particular ligand, the scoring function terms, and on the penalty for solvent binding. The occupancy state chromosome value is managed as a continuous variable between 0.0 and 1.0, with a nominal mutation step size of 1.0. Chromosome values lower than the occupancy threshold (defined as 1.0 - OCCUPANCY) result in the solvent being disabled; values higher than the threshold result in the solvent being enabled.

### Cavity mapping¶

The cavity mapping section is mandatory. You should choose one of the mapping algorithms shown below. All mapping parameters should be defined in SECTION MAPPER.

Table 14 Two sphere site mapping parameters

Parameter

Description

Type

Default

Range of values

Main user parameters

SITE_MAPPER

Mapping algorithm specifier

string literal

RbtSphereSiteMapper

fixed

CENTER

(x,y,z) center of cavity mapping region

Bracketed cartesian coordinate string (x,y,z)

None

None

RADIUS

Radius of cavity mapping region

float (Angstroms)

10.0

>0.0 (10.0–20.0 suggested range)

SMALL_SPHERE

Radius of small probe

float (Angstroms)

1.5

>0.0 (1.0–2.0 suggested range)

LARGE_SPHERE

Radius of large probe

float (Angstroms)

4.0

>SMALL_SPHERE (3.5–6.0 suggested range)

MAX_CAVITIES

Maximum number of cavities to accept (in descending order of size)

integer

99

>0

Advanced parameters (less frequently changed by the user)

VOL_INCR

Receptor atom radius increment for excluded volume

float (Angstroms)

0.0

>=0.0

GRID_STEP

Grid resolution for mapping

float (Angstroms)

0.5

>0.0 (0.3–0.8 suggested range)

MIN_VOLUME

Minimum cavity volume to accept (in Å3, not grid points)

float (Angstroms3)

100

>0 (100–300 suggested range)

Table 15 Reference ligand site mapping parameters

Parameter

Description

Type

Default

Range of values

Main user parameters

SITE_MAPPER

Mapping algorithm specifier

string literal

RbtLigandSiteMapper

fixed

REF_MOL

Reference ligand SD file name

string

ref.sd

None

RADIUS

Radius of cavity mapping region

float (Angstroms)

10.0

>0.0 (10.0–20.0 suggested range)

SMALL_SPHERE

Radius of small probe

float (Angstroms)

1.5

>0.0 (1.0–2.0 suggested range)

LARGE_SPHERE

Radius of large probe

float (Angstroms)

4.0

>SMALL_SPHERE (3.5–6.0 suggested range)

MAX_CAVITIES

Maximum number of cavities to accept (in descending order of size)

integer

99

>0

Advanced parameters (less frequently changed by the user)

VOL_INCR

Receptor atom radius increment for excluded volume

float (Angstroms)

0.0

>=0.0

GRID_STEP

Grid resolution for mapping

float (Angstroms)

0.5

>0.0 (0.3–0.8 suggested range)

MIN_VOLUME

Minimum cavity volume to accept (in Å3, not grid points)

float (Å3)

100

>0 (100–300 suggested range)

### Cavity restraint¶

The cavity restraint penalty function is mandatory and is designed to prevent the ligand from exiting the docking site. The function is calculated over all non-hydrogen atoms in the ligand (and over all explicit water oxygens that can translate). The distance from each atom to the nearest cavity grid point is calculated. If the distance exceeds the value of RMAX, a penalty is imposed based on the value of (distance - RMAX). The penalty can be either linear or quadratic depending on the value of the QUADRATIC parameter. It should not be necessary to change any the parameters in this section. Note that the docking protocol itself will manipulate the WEIGHT parameter, so any changes made to WEIGHT will have no effect.

SECTION CAVITY
SCORING_FUNCTION RbtCavityGridSF
WEIGHT 1.0
RMAX 0.1
END_SECTION


### Pharmacophore restraints¶

This section need only be defined if you wish to dock with pharmacophore restraints. If you are running conventional free docking then this section is not required. All pharmacophore definition parameters should be defined in SECTION PHARMA.

Table 16 Pharmacophore restraint parameters

Parameter

Description

Type

Default

Range of values

CONSTRAINTS_FILE

Mandatory pharmacophore restraints file

File name string

None (mandatory parameter)

Valid file name

OPTIONAL_FILE

Optional pharmacophore restraints file

File name string

None (optional parameter)

Valid file name, or empty

NOPT

Number of optional restraints that should be met

Integer

0

Between 0 and number of restraints in OPTIONAL_FILE

WRITE_ERRORS

Ligands with insufficient pharmacophore features to match the mandatory restraints are always removed prior to docking. If this parameter is true, the pre-filtered ligands are written to an error SD file with the same root name as the docked pose output SD file, but with an _errors.sd suffix. If false, the pre-filtered ligands are not written.

Boolean

FALSE

TRUE of FALSE

WEIGHT

Overall weight for the pharmacophore penalty function

Float

1.0

>=0.0

Calculation of mandatory restraint penalty The list of ligand atoms that matches each restraint type in the mandatory restraints file is precalculated for each ligand as it is loaded. If the ligand contains insufficient features to satisfy all of the mandatory restraints the ligand is rejected and is not docked. Note that the rejection is based purely on feature counts and does not take into account the possible geometric arrangements of the features. Rejected ligands are optionally written to an error SD file. The penalty for each restraint is based on the distance from the nearest matching ligand atom to the pharmacophore restraint centre. If the distance is less than the defined tolerance (restraint sphere radius), the penalty is zero. If the distance is greater than the defined tolerance a quadratic penalty is applied, equal to (nearest distance - tolerance)2.

Calculation of optional restraint penalty The individual restraint penalties for each restraint in the optional restraints file are calculated in the same way as for the mandatory penalties. However, only the NOPT lowest scoring (least penalised) restraints are summed for any given docking pose. Any remaining higher scoring optional restraints are ignored and do not contribute to the total pharmacophore restraint penalty.

Calculation of overall restraint penalty The overall pharmacophore restraint penalty is the sum of the mandatory restraint penalties and the NOPT lowest scoring optional restraint penalties, multiplied by the WEIGHT parameter value.

### NMR restraints¶

To be completed. However, this feature has rarely been used.

### Example system definition files¶

Full system definition file with all sections and common parameters enumerated explicitly:

RBT PARAMETER_FILE_V1.00
TITLE HSP90-PU3-lig-cavity, solvent flex=5
RECEPTOR_FILE PROT_W3_flex.mol2
RECEPTOR_SEGMENT_NAME PROT
RECEPTOR_FLEX 3.0
SECTION SOLVENT
FILE PROT_W3_flex_5.pdb
TRANS_MODE TETHERED
ROT_MODE TETHERED
MAX_TRANS 1.0
MAX_ROT 30.0
OCCUPANCY 0.5
END_SECTION
SECTION_LIGAND
TRANS_MODE FREE
ROT_MODE FREE
DIHEDRAL_MODE FREE
MAX_TRANS 1.0
MAX_ROT 30.0
MAX_DIHEDRAL 30.0
END_SECTION
SECTION MAPPER
SITE_MAPPER RbtLigandSiteMapper
REF_MOL ref.sd
SMALL_SPHERE 1.0
MIN_VOLUME 100
MAX_CAVITIES 1
VOL_INCR 0.0
GRIDSTEP 0.5
END_SECTION
SECTION CAVITY
SCORING_FUNCTION RbtCavityGridSF
WEIGHT 1.0
END_SECTION
SECTION PHARMA
SCORING_FUNCTION RbtPharmaSF
WEIGHT 1.0
CONSTRAINTS_FILE mandatory.const
OPTIONAL FILE optional.const
NOPT 3
WRITE_ERRORS TRUE
END_SECTION


## Molecular files and atoms typing¶

Macromolecular targets (protein or RNA) are input from Tripos MOL2 files (RbtMOL2FileSource class) or from pairs of Charmm PSF (RbtPsfFileSource class) and CRD (RbtCrdFileSource class) files. Ligands are input from MDL SD files (RbtMdlFileSource class). Explicit structural waters are input optionally from PDB files (RbtPdbFileSource class). Ligand docking poses are output to MDL SD files.

The rDock scoring functions have been defined and validated for implicit non-polar hydrogen (extended carbon) models only. If you provide all-atom models, be aware that the non-polar hydrogens will be removed automatically. Polar hydrogens must be defined explicitly in the molecular files, and are not added by rDock. Positive ionisable and negative ionisable groups can be automatically protonated and deprotonated respectively to create common charged groups such as guanidinium and carboxylic acid groups.

MOL2 is now the preferred file format for rDock as it eliminates many of the atom typing issues inherent in preparing and loading PSF files. The use of PSF/CRD files is strongly discouraged. The recommendation is to prepare an all-atom MOL2 file with correct Tripos atom types assigned, and allow rDock to remove non-polar hydrogens on-the-fly.

### Atomic properties¶

rDock requires the following properties to be defined per atom. Depending on the file format, these properties may be loaded directly from the molecular input file, or derived internally once the model is loaded:

• Cartesian (x,y,z) coordinates

• Element (atomic number)

• Formal hybridisation state (sp, sp2, sp3, aromatic, trigonal planar)

• Formal charge

• Distributed formal charge (known informally as group charge)

• Tripos force field type (rDock uses a modified version of the Sybyl 5.2 types, extended to include carbon types with implicit non-polar hydrogens)

• Atom name

• Substructure (residue) name

• Atomic radius (assigned per element from $RBT_ROOT/data/RbtElements.dat) Note The rDock scoring functions do not use partial charges and therefore partial charges do not have to be defined. The atomic radii are simplified radii defined per element, and are used for cavity mapping and in the polar scoring function term, but are not used in the vdW scoring function term. The latter has its own indepedent parameterisation based on the Tripos force field types. ### Difference between formal charge and distributed formal charge¶ The formal charge on an atom is always an integer. For example, a charged carboxylic acid group (COO-) can be defined formally as a formal double bond to a neutral oxygen sp2, and a formal single bond to a formally charged oxygen sp3. In reality of course, both oxygens are equivalent. rDock distributes the integer formal charge across all equivalent atoms in the charged group that are topologically equivalent. In negatively charged acid groups, the formal charge is distributed equally between the acid oxygens. In positively charged amines, the formal charge is distributed equally between the hydrogens. In charged guanidinium, amidinium, and imidazole groups, the central carbon also receives an equal portion of the formal charge (in addition to the hydrogens). The distributed formal charge is also known as the group charge. The polar scoring functions in rDock use the distributed formal charge to scale the polar interaction strength of the polar interactions. ### Parsing a MOL2 file¶ MOLECULE, ATOM, BOND and SUBSTRUCTURE records are parsed. The atom name, substructure name, Cartesian coordinates and Tripos atom type are read directly for each atom. The element type (atomic number) and formal hybridisation state are derived from the Tripos type using an internal lookup table. Formal charges are not read from the MOL2 file and do not have to be assigned correctly in the file. Distributed formal charges are assigned directly by rDock based on standard substructure and atom names as described below. ### Parsing an SD file¶ Cartesian coordinates, element and formal charge are read directly for each atom. Formal bond orders are read for each bond. Atom names are derived from element name and atom ID (e.g. C1, N2, C3). The substructure name is MOL. Formal hybridisation states are derived internally for each atom based on connectivity patterns and formal bond orders. The Tripos types are asssigned using internal rules based on atomic number, formal hybridisation state and formal charges. The integer formal charges are distributed automatically across all topologically equivalent atoms in the charged group. ### Assigning distributed formal charges to the receptor¶ rDock provides a file format independent method for assigning distributed formal charges directly to the receptor atoms, which is used by the MOL2 and PSF/CRD file readers. The method uses a lookup table based on standard substructure and atom names, and does not require the integer formal charges to be assigned to operate correctly. The lookup table file is $RBT_ROOT/data/sf/RbtIonicAtoms.prm. Each section name represents a substructure name that contains formally charged atoms. The entries within the section represent the atom names and distributed formal charges for that substructure name. The file provided with rDock contains entries for all standard amino acids and nucleic acids, common metals, and specific entries required for processing the GOLD CCDC/Astex validation sets.

Important You may have to extend RbtIonicAtoms.prm if you are working with non-standard receptor substructure names and/or atom names, in order for the distributed formal charges to be assigned correctly.

## rDock file formats¶

### .prm file format¶

The .prm file format is an rDock-specific text format and is used for:

• system definition files (known previously as receptor .prm files)

• scoring function definition files

• search protocol definition files

The format is simple and allows for an arbitrary number of named parameter/value pairs to be defined, optionally divided into named sections. Sections provide a namespace for parameter names, to allow parameter names to be duplicated within different sections. The key features of the format are:

• The first line of the file must be RBT_PARAMETER_FILE_V1.00 with no preceeding whitespace.

• Subsequent lines may contain either:

1. comment lines

2. reserved keywords TITLE, SECTION, or END_SECTION

3. parameter name/value pairs

• Comment lines should start with a # character in the first column with no preceeding whitespace, and are ignored.

• The reserved words must start in the first column with no preceeding whitespace.

• The TITLE record should occur only once in the file and is used to provide a title string for display by various scripts such as run rbscreen.pl. The keyword should be followed by a single space character and then the title string, which may contain spaces. If the TITLE line occurs more than once, the last occurence is used.

• SECTION records can occur more than once, and should always be paired with a closing END_SECTION record. The keyword should be followed by a single space character and then the section name, which may NOT itself contain spaces. All section names must be unique within a .prm file. All parameter name/value pairs within the SECTION / END_SECTION block belong to that section.

• Parameter name/value pairs are read as free-format tokenised text and can have preceeding, trailing, and be separated by arbitrary whitespace. This implies that the parameter name and value strings themselves are not allowed to contain any spaces. The value strings are interpreted as numeric, string, or boolean values as appropriate for that parameter. Boolean values should be entered as TRUE or FALSE uppercase strings.

Caution The current implementation of the .prm file reader does not tolerate a TAB character immediately following the TITLE and SECTION keywords. It is very important that the first character after the SECTION keyword in particular is a true space character, otherwise the reserved word will not be detected and the parameters for that section will be ignored.

Example .prm file In the following example, RECEPTOR_FILE is defined in the top level namespace. The remaining parameters are defined in the MAPPER and CAVITY namespaces. The indentation is for readability, and has no significance in the format.

RBT_PARAMETER_FILE_V1.00
TITLE 4dfr oxido-reductase

RECEPTOR_FILE 4dfr.mol2

SECTION MAPPER
SITE_MAPPER RbtLigandSiteMapper
REF_MOL 4dfr_c.sd
SMALL_SPHERE 1.0
MIN_VOLUME 100
MAX_CAVITIES 1
VOL_INCR 0.0
GRIDSTEP 0.5
END_SECTION

SECTION CAVITY
SCORING_FUNCTION RbtCavityGridSF
WEIGHT 1.0
END_SECTION


### Water PDB file format¶

rDock requires explicit water PDB files to be in the style as output by the Dowser program. In particular:

• Records can be HETATM or ATOM

• The atom names must be OW, H1 and H2

• The atom records for each water molecule must belong to the same subunit ID

• The subunit IDs for different waters must be distinct, but do not have to be consecutive

• The atom IDs are not used and do not have to be consecutive (they can even be duplicated)

• The order of the atom records within a subunit is unimportant

• The temperature factor field of the water oxygens can be used to define the per-solvent flexibility modes. The temperature factors of the water hydrogens are not used.

Table 17 Conversion of temperature values to solvent flexibility modes

PDB temperature factor

Solvent translational flexibility

Solvent rotational flexibility

0

FIXED

FIXED

1

FIXED

TETHERED

2

FIXED

FREE

3

TETHERED

FIXED

4

TETHERED

TETHERED

5

TETHERED

FREE

6

FREE

FIXED

7

FREE

TETHERED

8

FREE

FREE

Example Valid rDock PDB file for explicit, flexible waters:

REMARK tmp 1YET.pdb xtal_hoh.pdb
HETATM 3540 OW HOH W 106 28.929 12.684 20.864 1.00 1.0
HETATM 3540 H1 HOH W 106 28.034 12.390 21.200 1.00
HETATM 3540 H2 HOH W 106 29.139 12.204 20.012 1.00
HETATM 3542 OW HOH W 108 27.127 14.068 22.571 1.00 2.0
HETATM 3542 H1 HOH W 108 26.632 13.344 23.052 1.00
HETATM 3542 H2 HOH W 108 27.636 13.673 21.806 1.00
HETATM 3679 OW HOH W 245 27.208 10.345 27.250 1.00 3.0
HETATM 3679 H1 HOH W 245 27.657 10.045 26.409 1.00
HETATM 3679 H2 HOH W 245 26.296 10.693 27.036 1.00
HETATM 3680 OW HOH W 246 31.737 12.425 21.110 1.00 4.0
HETATM 3680 H1 HOH W 246 31.831 12.448 22.106 1.00
HETATM 3680 H2 HOH W 246 30.775 12.535 20.863 1.00


### Pharmacophore restraints file format¶

Pharmacophore restraints are defined in a simple text file, with one restraint per line. Each line should contain the following values, separated by commas or whitespace:

x y z coords of restraint centre, tolerance (in Angstroms), restraint type string


The supported restraint types are:

Table 18 Pharmacophore restraint types

String

Description

Matches

Any

Any atom

Any non-hydrogen atom

Don

H-bond donor

Any neutral donor hydrogen

Acc

H-bond acceptor

Any neutral acceptor

Aro

Aromatic

Any aromatic ring centre (pseudo atom)

Hyd

Hydrophobic

Any non-polar hydrogens (if present), any C sp3 or S sp3, any C or S not bonded to O sp2, any Cl, Br, I

Hal

Hydrophobic, aliphatic

Subset of Hyd, sp3 atoms only

Har

Hydrophobic, aromatic

Subset of Hyd, aromatic atoms only

Ani

Anionic

Any atom with negative distributed formal charge

Cat

Cationic

Any atom with positive distributed formal charge

## rDock programs¶

Programs summary tables:

Table 19 Core rDock C++ executables

Executable

Used for

Description

rbcavity

Preparation

Cavity mapping and preparation of docking site (.as) file.

rbcalcgrid

Preparation

Calculation of vdW grid files (usually called by make_grid.csh wrapper script).

rbdock

Docking

The main rDock docking engine itself.

Table 20 Auxiliary rDock programs

Executable

Used for

Description

sdtether

Preparation

Prepares a ligand SD file for tethered scaffold docking. Annotates ligand SD file with tethered substructure atom indices. Requires Open Babel Python bindings.

rbhtfinder

Preparation

Used to optimise a high-throughput docking protocol from an initial exhaustive docking of a small representative ligand library. Parametrize a multi-step protocol for your system.

make_grid.csh

Preparation

Creates the vdW grid files required for grid-based docking protocols (dock_grid.prm and dock_solv_grid.prm). Simple front-end to rbcalcgrid.

rbconvgrid

Analysis

Converts rDock vdW grids to InsightII grid format for visualisation.

rbmoegrid

Analysis

Converts rDock vdW grids to MOE grid format for visualisation.

rblist

Analysis

Outputs miscellaneous information for ligand SD file records.

sdrmsd

Analysis

Calculation of ligand Root Mean Squared Displacement (RMSD) between reference and docked poses, taking into account ligand topological symmetry. Requires Open Babel Python bindings.

sdfilter

Analysis

Utility for filtering SD files by arbitrary data field expressions. Useful for simple post-docking filtering by score components.

sdsort

Analysis

Utility for sorting SD files by arbitrary data field. Useful for simple post-docking filtering by score components.

sdreport

Analysis

Utility for reporting SD file data field values. Output in tab-delimited or CSV format.

sdsplit

Utility

Splits an SD file into multiple smaller SD files of fixed number of records.

sdmodify

Utility

Sets the molecule title line of each SD record equal to a given SD data field.

### Programs reference¶

#### rbdock¶

rbdock - the rDock docking engine itself.

$RBT_ROOT/bin/rbdock {-i input ligand MDL SD file} {-o output MDL SD file} {-r system definition.prm file} {-p docking protocol.prm file} [-n number of docking runs/ligand] [-s random seed] [-T debug trace level] [[-t SCORE.INTER threshold] | [-t filter definition file]] [-ap -an -allH -cont]  Simple exhaustive docking The minimum requirement for rbdock is to specify the input (-i) and output (-o) ligand SD file names, the system definition .prm file (-r) and the docking protocol .prm file (-p). This will perform one docking run per ligand record in the input SD file and output all docked ligand poses to the output SD file. Use -n to increase the number of docking runs per ligand record. High-throughput docking 1. The -t and -cont options can be used to construct high-throughput protocols. If the argument following -t is numeric it is interpreted as a threshold value for SCORE.INTER, the total intermolecular score between ligand and receptor/solvent. In the absence of -cont, the threshold acts as an early termination filter, and the docking runs for each ligand will be terminated early once the threshold value has been exceeded. Note that the threshold is applied only at the end of each individual docking run, not during the runs themselves. If the -cont (continue) option is specified as well, the threshold acts as an output pose filter instead of a termination filter. The docking runs for each ligand run to completion as in the exhaustive case, but only the docking poses that exceed the threshold value of SCORE.INTER are written to the output SD file. High throughput docking 2. Alternatively, if the argument following -t is non-numeric it is interpreted as a filter definition file. The filter definition file can be used to define multiple termination filters and multiple output pose filters in a generic way. Any docking score component can be used in the filter definitions. run rbscreen.pl generates a filter definition file for multi-stage, highthroughput docking, with progressive score thresholds for early termination of poorly performing ligands. The use of filter definition files is preferred over the more limited SCORE.INTER filtering described above, whose use is now deprecated. Automated ligand protonation/deprotonation The -ap option activates the automated protonation of ligand positive ionisable centres, notably amines, guanidines, imidazoles, and amidines. The -an option activates the automated deprotonation of ligand negative ionisable centres, notably carboxylic acids, phosphates, phosphonates, sulphates, and sulphonates. The precise rules used by rDock for protonation and deprotonation are quite crude, and are not user-customisable. Therefore these flags are not recommended for detailed validation experiments, in which care should be taken that the ligand protonation states are set correctly in the input SD file. Note that rDock is not capable of converting ionised centres back to the neutral form; these are unidirectional transformations. Control of ligand non-polar hydrogens By default, rDock uses an implicit non-polar hydrogen model for receptor and ligand, and all of the scoring function validation has been performed on this basis. If the -allH option is not defined (recommended), all explicit non-polar hydrogens encountered in the ligand input SD file are removed, and only the polar hydrogens (bonded to O, N, or S) are retained. If the -allH option is defined (not recommended), no hydrogens are removed from the ligand. Note that rDock is not capable of adding explicit non-polar hydrogens, if none exist. In other words, the -allH option disables hydrogen removal, it does not activate hydrogen addition. You should always make sure that polar hydrogens are defined explicitly. If the ligand input SD file contains no explicit non-polar hydrogens, the -allH option has no effect. Receptor protonation is controlled by the system definition prm file. #### rbcavity¶ rbcavity - Cavity mapping and preparation of docking site (.as) file. $RBT_ROOT/bin/rbcavity
{-r system definition .prm file}
[-ras -was -d -v -s]
[-l distance from cavity]
[-b border]


Exploration of cavity mapping parameters rbcavity - r.prmf file You can run rbcavity with just the -r argument when first preparing a new receptor for docking. This allows you to explore rapidly the impact of the cavity mapping parameters on the generated cavities, whilst avoiding the overhead of actually writing the docking site (.as) file to disk. The number of cavities and volume of each cavity are written to standard output.

Visualisation of cavities rbcavity - r.prmf file - d If you have access to InsightII you can use the -d option to dump the cavity volumes in InsightII grid file format. There is no need to write the docking site (.as) file first. The InsightII grid files should be loaded into the reference coordinate space of the receptor and contoured at a contour level of 0.99.

Writing the docking site (.as) file rbcavity - r.prmf file - was When you are happy the mapping parameters, use the -was option to write the docking site (.as) file to disk. The docking site file is a binary file that contains the cavity volumes in a compact format, and a pre-calculated cuboid grid extending over the cavities. The grid represents the distance from each point in space to the nearest cavity grid point, and is used by the cavity penalty scoring function. Calculating the distance grid can take a long time (whereas the cavity mapping itself is usually very fast), hence the -was option should be used sparingly.

Analysis of cavity atoms rbcavity - r.prmf file -ras -ldistance Use the -l options to list the receptor atoms within a given distance of any of the cavity volumes, for example to determine which receptor OH/NH3+ groups should be flexible. This option requires access to the pre-calculated distance grid embedded within the docking site (.as) file, and is best used in combination with the -ras option, which loads a previously generated docking site file. This avoids the time consuming step of generating the cavity distance grid again. If -l is used without -ras, the cavity distance grid will be calculated on-the-fly each time.

Miscellaneous options The -s option writes out various statistics on the cavity and on the receptor atoms in the vicinity of the cavity. These values have been used in genetic programming model building for docking pose false positive removal. The -v option writes out the receptor coordinates in PSF/CRD format for use by the rDock Viewer (not documented here). Note that the PSF/CRD files are not suitable for simulation purposes, only for visualisation, as the atom types are not set correctly. The -b option controls the size of the cavity distance grid, and represents the border beyond the actual cavity volumes. It should not be necessary to vary this parameter (default = 8A) unless longer-range scoring functions are implemented.

#### rbcalcgrid¶

rbcalcgrid - Calculation of vdW grid files (usually called by make grid.csh wrapper script).

$RBT_ROOT/bin/rbcalcgrid {-r system definition file} {-o output suffix for generated grids} {-p vdW scoring function prm file} [-g grid step] [-b border]  Note that, unlike rbdock and rbcavity, spaces are not tolerated between the command-line options and their corresponding arguments. See $RBT_ROOT/bin/make_grid.csh for common usage.

#### sdrmsd¶

sdrmsd - Calculation of ligand Root Mean Squared Displacement (RMSD) between reference and docked poses. It takes into account molecule topological symmetry. Requires Open Babel Python bindings.

$RBT_ROOT/bin/sdrmsd [options] {reference SD file} {input SD file}  With two arguments sdrmsd calculates the RMSD between each record in the input SD file and the first record of the reference SD file. If there is a mismatch in the number of atoms, the record is skipped and the RMSD is not calculated. The RMSD is calculated over the heavy (non-hydrogen) atoms only. Results are output to standard output. If some record was skipped, a warning message will be printed to standard error. With fitting A molecular superposition will be done before calculation of the RMSD. The output will specify an RMSD FIT calculation was done. sdrmsd -o output.sdf reference.sdf input.sdf sdrmsd --out=output.sdf reference.sdf input.sdf  #### sdtether¶ sdtether - Prepares a ligand SD file for tethered scaffold docking. Requires Open Babel Python bindings. Annotates ligand SD file with tethered substructure atom indices. $RBT_ROOT/bin/sdtether {ref. SDfile} {in SDfile} {out SDfile} "{SMARTS query}"


sdtehter performs the following actions:

• Runs the SMARTS query against the reference SD file to determine the tethered substructure atom indices and coordinates.

• If more than one substructure match is retrieved (e.g. due to topological symmetry, or if the query is too simple) all substructure matchs are retained as the reference and all ligands will be tethered according to all possible matchs.

• Runs the SMARTS query against each record of the input ligand SD file in turn.

• For each substructure match, the ligand coordinates are transformed such that the principal axes of the matching substructure coordinates are aligned with the reference substructure coordinates.

• In addition, an SD data field is added to the ligand record which lists the atom indices of the substructure match, for later retrieval by rDock.

• Each transformed ligand is written to the output SD file.

• Note that if the SMARTS query returns more than one substructure match for a ligand, that ligand is written multiple times to the output file, once for each match, each of which will be docked independently with different tethering information

#### sdfilter¶

sdfilter - Post-process an SD file by filtering the records according to data fields or attributes.

Usage:

sdfilter -f '\$<DataField> <Operator> <Value >' [-s <DataField>] [sdFiles]


or

sdfilter -f <filename> [-s <DataField >] [sdFiles]


Notes Multiple filters are allowed and are OR’d together. Filters can be provided in a file, one per line. Standard Perl operators should be used. e.g.

eq ne lt gt le ge for strings
== != < > <= >= for numeric


_REC (record #) is provided as a pseudo-data field. If -s option is used, _COUNT (#occurrences of DataField) is provided as a pseudo-data field. If SD file list not given, reads from standard input. Output is to standard output.

For examples, read section EXAMPLES SECTION.

#### sdreport¶

sdreport - Produces text summaries of SD records

sdreport [-l] [-t [<FieldName, FieldName...>]] [-c <FieldName, FieldName...>] [-id <IDField>] [-nh] [-o] [-s] [-sup] [sdFiles]

-l (list format) output all data fields for each record as processed
-t (tab format) tabulate selected fields for each record as processed
-c (csv format) comma delimited output of selected fields for each record as processed
-s (summary format) output summary statistics for each unique value of ligand ID
-sup (supplier format) tabulate supplier details (from Catalyst)
-id <IDField> data field to use as ligand ID
-nh don't output column headings in -t and -c formats
-o use old (v3.00) score field names as default columns in -t and -c formats, else use v4.00 field names
-norm use normalised score filed names as default columns in -t and -c formats (normalised = score / #ligand heavy atoms)


Notes If -l, -t or -c are combined with -s, the listing/table is output withing each ligand summary. -sup should not be combined with other options. Default field names for -t and -c are RiboDock score field names. Default ID field name is Name. If sdFiles not given, reads from standard input. Output is to standard output.

#### sdsplit¶

sdsplit - Splits SD records into multiple files of equal size

sdsplit [-<RecSize>] [-o <OutputRoot>] [sdFiles]

-<RecSize> record size to split into (default = 1000 records)
-o <OutputRoot> Root name for output files (default = tmp)


If SD file list not given, reads from standard input.

#### sdsort¶

sdsort - sorts SD records by given data field

sdsort [-n] [-r] [-f <DataField>] [sdFiles]

-n numeric sort (default is text sort)
-r descending sort (default is ascending sort)
-f <DataField> specifies sort field
-s fast mode. Sorts the records for each named compound independently (must be consecutive)
-id <NameField> specifies compound name field (default = 1st title line)


Notes _REC (record #) is provided as a pseudo-data field. If SD file list not given, reads from standard input. Output is to standard output. Fast mode can be safely used for partial sorting of huge SD files of raw docking hits without running into memory problems.

#### rbmoegrid¶

rbmoegrid - calculates grids for a given atom type

rbmoegrid -o <OutputRoot> -r <ReceptorPrmFile> -p <SFPrmFile> [-g <GridStep> -b <border> -t <tripos_type>]

-o <OutFileName> (.grd is suffiexed)
-r <ReceptorPrmFile> - receptor param file (contains active site params)
-p <SFPrmFile> - scoring function param file (default calcgrid_vdw.prm)
-g <GridStep> - grid step (default = 0.5A)
-b <Border> - grid border around docking site (default = 1.0A)
-t <AtomType> - Tripos atom type (default is C.3)


#### rblist¶

rblist - output interaction center info for ligands in SD file (with optional autoionisation)

rblist -i <InputSDFile> [-o <OutputSDFile>] [-ap ] [-an] [-allH]

-i <InputSDFile> - input ligand SD file
-o <OutputSDFilde> - output SD file with descriptors (default = no output)
-ap - protonate all neutral amines, guanidines, imidazoles (default = disabled)
-an - deprotonate all carboxylic, sulphur and phosphorous acid groups (default = disabled)
-allH - read all hydrogens present (default = polar hydrogens only)
-tr - rotate all secondary amides to trans (default = leave alone)
-l - verbose listing of ligand atoms and rotable bonds (default = compact table format)


## Appendix¶

Table 21 Van der Waals parameters in Tripos 5.2 force field (R = radius (Å); K = well depth (kcal/mol); IP = Ionization potential (eV); POL = polarisability (1025 cm3)).

Atom Type

R

K

IP

POL

Description

H

1.5

0.042

13.6

4

Non-polar hydrogen

H.P

1.2

0.042

13.6

4

Polar hydrogen

C.3

1.7

0.107

14.61

13.8

C sp3 (0 implicit H)

C.3.H1

1.8

0.107

14.61

16.38

C sp3 (1 implicit H)

C.3.H2

1.9

0.107

14.61

19.27

C sp3 (2 implicit H)

C.3.H3

2

0.107

14.61

22.47

C sp3 (3 implicit H)

C.2

1.7

0.107

15.62

13.8

C sp2 (0 implicit H)

C.cat

1.7

0.107

15.62

13.8

C sp2 (guanidinium centre)

C.2.H1

1.8

0.107

15.62

16.38

C sp2 (1 implicit H)

C.2.H2

1.9

0.107

15.62

19.27

C sp2 (2 implicit H)

C.ar

1.7

0.107

15.62

13.8

C aromatic (0 implicit H)

C.ar.H1

1.8

0.107

15.62

16.38

C aromatic (1 implicit H)

C.1

1.7

0.107

17.47

13.8

C sp (0 implicit H)

C.1.H1

1.8

0.107

17.47

16.38

C sp (1 implicit H)

N.4

1.55

0.095

33.29

8.4

N sp3+ (cationic)

N.3

1.55

0.095

18.93

8.4

N sp3

N.pl3

1.55

0.095

19.72

8.4

N trigonal planar (non-amide)

N.am

1.55

0.095

19.72

8.4

N trigonal planar (amide)

N.2

1.55

0.095

22.1

8.4

N sp2

N.ar

1.55

0.095

22.1

8.4

N aromatic

N.1

1.55

0.095

23.91

8.4

N sp

O.3

1.52

0.116

24.39

5.4

O sp3

O.2

1.52

0.116

26.65

5.4

O sp2

O.co2

1.52

0.116

35.12

5.4

O carboxylate

S.3

1.8

0.314

15.5

29.4

S sp3

S.o

1.7

0.314

15.5

29.4

sulfoxide

S.o2

1.7

0.314

15.5

29.4

sulfone

S.2

1.8

0.314

17.78

29.4

S sp2

P.3

1.8

0.314

16.78

40.6

F

1.47

0.109

20.86

3.7

Cl

1.75

0.314

15.03

21.8

Br

1.85

0.434

13.1

31.2

I

1.98

0.623

12.67

49

Na

1.2

0.4

K

1.2

0.4

UNDEFINED

1.2

0.042

Table 22 Geometrical parameters for empirical terms (a = Geometric variable; b = Ideal value; c = Tolerance on ideal value; d = Deviation at which score is reduced to zero).

Term

$$X$$a

$$X_0$$b

$$X_{\min}$$c

$$X_{\max}$$d

Description

$$S_{\text{polar}}$$

$$R_{12}$$

$$R$$ + 0.05 Å

0.25 Å

0.6 Å

Distance between interaction centres

$$\alpha_{\text{DON}}$$

180°

30°

80°

Angle around donor H

$$\alpha_{\text{ACC}}$$

180°

60°

100°

Angle around acceptor

$$\alpha_{\text{C+}}$$

180°

60°

100°

Angle between C+ACC vector and normal to plane of guanidinium group

$$\phi_{\text{ACC}\_\text{LP}}$$

45°

15°

15°

From [RiboDock2004] Figure 2.

$$\theta_{\text{ACC}\_\text{LP}}$$

20°

60°

From [RiboDock2004] Figure 2.

$$\phi_{\text{ACC}\_\text{PLANE}}$$

60°

75°

From [RiboDock2004] Figure 2.

$$\theta_{\text{ACC}\_\text{PLANE}}$$

20°

60°

From [RiboDock2004] Figure 2.

$$S_{\text{repul}}$$

$$R_{12}$$

R + 1.1 Å

0.25 Å

0.6 Å

Distance between interaction centres

$$\alpha_{\text{DON}}$$

180°

30°

60°

Angle around donor H

$$\alpha_{\text{ACC}}$$

180°

30°

60°

Angle around acceptor

$$S_{\text{arom}}$$

$$R_{\text{perp}}$$

3.5Å

0.25 Å

0.6 Å

From [RiboDock2004] Figure 3.

$$\alpha_{\text{slip}}$$

20°

60 °

From [RiboDock2004] Figure 3.

Table 23 Angular functions used to describe attractive and repulsive polar interactions (a = Interaction centre types; b = angular functions in Equations 6-13 TODO).

$$\text{IC1}$$a

$$\text{ANG}_{\text{IC1}}$$b

$$\text{IC2}$$a

$$\text{ANG}_{\text{IC2}}$$b

Attractive ($$S_{\text{polar}}$$)

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

ACC_LP

$$f_1(|\Delta\phi_{\text{ACC}\_\text{LP}}|) \cdot f_1(|\Delta\theta_{\text{ACC}\_\text{LP}}|)$$

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

ACC_PLANE

$$f_1(|\Delta\phi_{\text{ACC}\_\text{PLANE}}|) \cdot f_1(|\Delta\theta_{\text{ACC}\_\text{PLANE}}|)$$

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

ACC

$$f_1(|\Delta\alpha_{\text{ACC}}|)$$

M+

1

ACC_LP

$$f_1(|\Delta\phi_{\text{ACC}\_\text{LP}}|) \cdot f_1(|\Delta\theta_{\text{ACC}\_\text{LP}}|)$$

M+

1

ACC_PLANE

$$f_1(|\Delta\phi_{\text{ACC}\_\text{PLANE}}|) \cdot f_1(|\Delta\theta_{\text{ACC}\_\text{PLANE}}|)$$

M+

1

ACC

$$f_1(|\Delta\alpha_{\text{ACC}}|)$$

1

ACC_LP

C+

$$f_1(|\Delta\alpha_{\text{C+}}|)$$

ACC_PLANE

$$f_1(|\Delta\alpha_{\text{ACC}}|)$$

1

ACC

Repulsive ($$S_{\text{repul}}$$)

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

M+

1

DON

$$f_1(|\Delta\alpha_{\text{DON}}|)$$

C+

1

M+

1

C+

1

C+

1

C+

1

ACC_LP

ACC_LP

ACC_PLANE

$$f_1(|\Delta\alpha_{\text{ACC}}|)$$

ACC_PLANE

$$f_1(|\Delta\alpha_{\text{ACC}}|)$$

ACC

ACC

Table 24 Solvation parameters (a = Frequency of occurrence in training set).

Atom type

Description

$$N$$a

$$r_i$$

$$p_i$$

$$w_i$$

C_sp3

Apolar carbon sp3 with 0 implicit H

48

1.7

2.149

0.8438

CH_sp3

Apolar carbon sp3 with 1 implicit H

59

1.8

1.276

0.0114

CH2_sp3

Apolar carbon sp3 with 2 implicit H

487

1.9

1.045

0.0046

CH3_sp3

Apolar carbon sp3 with 3 implicit H

409

2

0.88

0.0064

C_sp2

Apolar carbon sp2 with 0 implicit H

10

1.72

1.554

0.0789

CH_sp2

Apolar carbon sp2 with 1 implicit H

45

1.8

1.073

-0.0014

CH2_sp2

Apolar carbon sp2 with 2 implicit H

26

1.8

0.961

0.0095

C_sp2p

Positive charged carbon sp2

2

1.72

1.554

-0.7919

C_ar

Apolar aromatic carbon with 0 implicit H

116

1.72

1.554

0.017

CH_ar

Apolar aromatic carbon with 1 implicit H

357

1.8

1.073

-0.0143

C_sp

Carbon sp

24

1.78

0.737

-0.0052

C_sp3_P

Polar carbon sp3 with 0 implicit H

6

1.7

2.149

-0.0473

CH_sp3_P

Polar carbon sp3 with 1 implicit H

22

1.8

1.276

-0.0394

CH2_sp3_P

Polar carbon sp3 with 2 implicit H

130

1.9

1.045

-0.0078

CH3_sp3_P

Polar carbon sp3 with 3 implicit H

69

2

0.88

0.0033

C_sp2_P

Polar carbon sp2 with 0 implicit H

57

1.72

1.554

-0.2609

CH_sp2_P

Polar carbon sp2 with 1 implicit H

30

1.8

0.961

-0.005

CH2_sp2_P

Polar carbon sp2 with 2 implicit H

1

1.8

0.961

0.0095

C_ar_P

Polar aromatic carbon with 0 implicit H

53

1.72

1.554

-0.2609

CH_ar_P

Polar aromatic carbon with 1 implicit H

34

1.8

1.073

-0.0015

H

Explicit apolar hydrogen (not used)

0

1.2

1

0

HO

Polar hydrogen bonded to O

54

1

0.944

0.0499

HN

Polar hydrogen bonded to N

54

1.1

1.128

-0.0242

HNp

Positively charged polar hydrogen bonded to N

23

1.2

1.049

-1.9513

HS

Polar hydrogen bonded to S

4

1.2

0.928

0.0487

O_sp3

Ether oxygen

31

1.52

1.08

-0.138

OH_sp3

Alcohol/phenol oxygen

48

1.52

1.08

-0.272

O_tri

Ester oxygen

59

1.52

1.08

0.0965

OH_tri

Acid oxygen (neutral)

6

1.52

1.08

-0.0985

O_sp2

Oxygen sp2

83

1.5

0.926

-0.1122

ON

Nitro group oxygen

18

1.5

0.926

-0.0055

Om

Negatively charged oxygen (carboxylate etc.)

7

1.7

0.922

-0.717

N_sp3

Nitrogen sp3 with 0 attached H

8

1.6

1.215

-0.6249

NH_sp3

Nitrogen sp3 with 1 attached H

11

1.6

1.215

-0.396

NH2_sp3

Nitrogen sp3 with 2 attached H

11

1.6

1.215

-0.215

N_sp3p

Nitrogen sp3+

6

1.6

1.215

-0.1186

N_tri

Amide nitrogen with 0 attached H

15

1.55

1.028

-0.23

NH_tri

Amide nitrogen with 1 attached H

8

1.55

1.028

-0.4149

NH2_tri

Amide nitrogen with 2 attached H

6

1.55

1.028

-0.1943

N_sp2

Nitrogen sp2

3

1.55

1.413

-0.0768

N_sp2p

Nitrogen sp2+

5

1.55

1.413

-0.2744

N_ar

Aromatic nitrogen

26

1.55

1.413

-0.531

N_sp

Nitrogen sp

6

1.55

1

-0.1208

S_sp3

Sulphur sp3

15

1.8

1.121

-0.0685

S_sp2

Sulphur sp2

5

1.8

1.121

-0.0314

P

Phosphorous

10

1.8

1.589

-1.275

F

Fluorine

99

1.47

0.906

0.0043

Cl

Chlorine

132

1.75

0.906

-0.0096

Br

Bromine

37

1.85

0.898

-0.0194

I

Iodine

9

1.98

0.876

-0.0189

Metal

All metals

0

0.7

1

-1.6667

UNDEFINED

Undefined types

0

1.2

1

0