This section does not pretend to be a comprehensive user guide. It does, however, highlight the key steps the user must take for different docking strategies, and may serve as a useful checklist in writing such a guide in the future.
By standard docking, we refer to docking of a flexible, untethered ligand to a receptor in the absence of explicit structural waters or any experimental restraints.
Standard docking workflow¶
Prepare a MOL2 file for the protein or nucleic acid target, taking into account the atom typing issues described above for MOL2 file parsing. The recommendation is to prepare an all-atom MOL2 file and allow RxDock to remove the non-polar hydrogens on-the-fly.
Make sure that any non-standard atom names and substructure names are defined in
$RBT_ROOT/data/sf/RbtIonicAtoms.prmin order for the assignment of distributed formal charges to work correctly. Make sure that the Tripos atom types are set correctly. RxDock uses the Tripos types to derive other critical atomic properties such as atomic number and hybridisation state.
The RxDock MOL2 parser was developed to read the CCDC/Astex protein
.mol2files, therefore this validation set is the de facto standard reference. You should compare against the format of the CCDC/Astex MOL2 files if you are in doubt as to whether a particular MOL2 file is suitable for RxDock.
Prepare a system definition file. At a minimum, you need to define the receptor parameters, the cavity mapping parameters (
SECTION MAPPER) and the cavity restraint penalty (
SECTION CAVITY). Make sure you define the
RECEPTOR_FLEXparameter if you wish to activate sampling of terminal OH and NH3+ groups in the vicinity of the docking site.
Generate the docking site (.as) file using
rbcavity. You will require a reference bound ligand structure in the coordinate space of the receptor if you wish to use the reference ligand cavity mapping method.
Prepare the ligand SD files you wish to dock, taking into account the atom typing issues described above for SD file parsing. In particular, make sure that formal charges and formal bond order are defined coherently so that there are no formal valence errors in the file. RxDock will report any perceived valence errors but will dock the structures anyway. Note that RxDock never samples bond lengths, bond angles, ring conformations, or non-rotatable bonds during docking so initial conformations should be reasonable.
Run a small test calculation to check that the system is defined correctly. For example, run
rbdockfrom the command line with a small ligand SD file, with the score-only protocol (
-p score.prm) and with the
-T 2option to generate verbose output. The output will include receptor atom properties, ligand atom properties, flexibility parameters, scoring function parameters and docking protocol parameters.
When satisfied, launch the full-scale calculations. A description of the various means of launching RxDock is beyond the scope of this guide.
Tethered scaffold docking¶
In tethered scaffold docking, the ligand poses are restricted and forced to overlay the substructure coordinates of a reference ligand. The procedure is largely as for standard docking, except that:
Ligand SD files must be prepared with the
rbtetherutility to annotate each record with the matching substructure atom indices, and to transform the coordinates of each ligand so that the matching substructure coordinates are overlaid with the reference substructure coordinates. This requires a Daylight SMARTS toolkit license.
The system definition file should contain a
SECTION LIGANDto define which of the the ligand degrees of freedom should be tethering to their reference values. Tethering can be applied to position, orientation and dihedral degrees of freedom independently. Note that the tethers are applied directly within the chromosome representation used by the search engine (where they affect the randomisation and mutation operators), and therefore external restraint penalty functions to enforce the tethers are not required.
The reference state values for each tethered degree of freedom are defined
directly from the initial conformation of each ligand as read from the input
SD file, and not from the reference SD file used by
rbtether. This is
why the ligand coordinates are transformed by
rbtether, such that each
ligand record can act as its own reference state. The reference SD file used
rbtether is not referred to by the docking calculation itself.
It follows from the above that tethered ligand docking is inappropriate for
input ligand SD files that have not already been transformed to the coordinate
space of the docking site, either by
rbtether or by some other means.
Example ligand definition for tethered scaffold¶
This definition will tether the position and orientation of the tethered substructure, but will allow free sampling of ligand dihedrals.
SECTION LIGAND TRANS_MODE TETHERED ROT_MODE TETHERED DIHEDRAL_MODE FREE MAX_TRANS 1.0 MAX_ROT 30.0 END_SECTION
Docking with pharmacophore restraints¶
In pharmacophore restrained docking, ligand poses are biased to fit user-defined pharmacophore points. The bias is introduced through the use of an external penalty restraint, which penalises docking poses that do not match the pharmacophore restraints. Unlike tethered scaffold docking, there is no modification to the chromosome operators themselves, hence the search can be inefficient, particularly for large numbers of restraints and/or for ligands with large numbers of matching features. Pre-screening of ligands is based purely on feature counts, and not on geometric match considerations.
The implementation supports both mandatory and optional pharmacophore
restraints. The penalty function is calculated over all mandatory restraints,
and over (any
NOPT from N) of the optional restraints. For example, you may
wish to ensure that any 4 from 7 optional restraints are satisfied in the
The procedure is largely as for standard docking, except that:
You should prepare separate pharmacophore restraint files for the mandatory and optional restraints. Note that optional restraints do not have to be defined, it is sufficient to only define at least one mandatory restraint.
The system definition file should contain a
SECTION PHARMAto add the pharmacophore restraint penalty to the scoring function.
Docking with explicit waters¶
Explicit structural waters can be loaded from an external PDB file,
independently from the main receptor model, by adding a
to the system definition file. The user has fine control over the flexibility of
each water molecule. A total of 9 flexibility modes are possible, in which the
translational and rotational degrees of freedom of each water can be set
FREE. Thus, for example, it is
possible to define a water with a fixed oxygen coordinate (presumably at a
crystallographically observed position), but freely rotating such that the
orientation of the water hydrogens can be optimised by the search engine (and
can be ligand-dependent).
In the current implementation, solvent refers strictly to water molecules, and the format of the water PDB file is very strictly defined. In future implementations it is anticipated that other, larger (and possibly flexible) molecules will be loadable as solvent, and that other file formats will be supported.
Explicit waters workflow¶
Prepare a separate PDB file for the explicit waters according to the format prescribed (the section called Water PDB file format).
SECTION SOLVENTto the system definition file and define the relevant flexibility parameters (Table 13). The minimal requirement is to define the
Decide whether you wish to have different per-solvent flexibility modes (defined via the occupancy values and temperature factor values in the PDB file (Table 17)), or whether you wish to have a single flexibility mode applied to all waters (defined via the
ROT_MODEvalues in the
SECTION SOLVENTof the receptor .prm file).
If you wish to use per-solvent flexibility modes (that is, you wish to set different modes for different waters) make sure that you do not define
ROT_MODEentries in the
SECTION SOLVENTas these values will override the per-solvent values derived from the temperature factors in the PDB file.
If you have defined any waters with
TETHEREDtranslational or rotational degrees of freedom, define
MAX_ROTvalues as appropriate (or accept the default values). The tethered ranges are applied to all tethered waters and can not be defined on a per-solvent basis at present.