How to Find a Binding Site for Molecular Docking When No Reference Ligand Is Available

This guide is for researchers starting a small-molecule docking study without a usable co-crystallized ligand. Its purpose is to help choose a defensible site hypothesis, or recognize when the evidence is too weak to proceed. Box dimensions and sampling settings come afterward.

“No ligand in this structure” can describe several different situations.

Do not discard useful site evidence simply because the chosen coordinate file is ligand-free. Conversely, the absence of a co-crystal does not justify treating every surface cavity as an equally credible target.

If receptor identity or state is unresolved, first use the holo, apo and AlphaFold receptor-selection guide.

State the intended mechanism in one or two sentences. Are you investigating an orthosteric site, a candidate allosteric region, a protein interface or another specific interaction?

Then identify what evidence would support that location:

• An experimental structure with a relevant ligand, substrate or cofactor in a related system.
• A credible mapping of functional residues.
• Biochemical evidence that constrains the region.
• Structural conservation compatible with the proposed mechanism.
• A cavity prediction that remains plausible in the biological assembly.

Keep limitations beside the evidence. A mutation affecting activity does not, by itself, show that the mutated residue contacts the ligand. A homologous fold does not establish conserved selectivity. A predicted cavity does not establish functional relevance.

This is a recommended evidence hierarchy for planning a study, not a numeric validation score. Its purpose is to prevent docking results from becoming the sole justification for the site in which those results were generated.

The two methods provide different ways to inspect a structure.

P2Rank evaluates local surface environments using a trained model, then groups favorable regions into predicted sites. Fpocket detects and groups geometric features derived from alpha spheres to identify candidate cavities. Their original papers explain these different representations and evaluation methods. Krivák and Hoksza, P2Rank, Le Guilloux, Schmidtke and Tufféry, fpocket.

Neither output should be treated as a ligand-specific binding measurement.

A reproducible candidate-generation record should include the exact input structure, selected chains and model, preparation decisions, tool version, model or configuration, command, output files and any warnings.

For a concrete output contract, the P2Rank 2.5.1 documentation describes ranked pocket records with scores, center coordinates, adjacent residues and atoms, and a calibrated binding-site probability. Use the documentation matching the version actually installed; do not mix stable output fields with an unreleased development example.

The highest-ranked pocket is the method's preferred candidate under that input and configuration. A calibrated pocket probability is not the probability that a particular compound binds, that an allosteric mechanism exists, or that the target is therapeutically useful.

Do not add P2Rank and fpocket scores together or compare their raw values as if they were on a common scale. Instead, ask whether the methods point to the same physical region and what independent evidence supports that region.

Agreement can strengthen a hypothesis, but it is not two independent experiments: both predictions depend on the supplied structure. Disagreement is also informative. It may expose differences in representation, pocket boundaries or sensitivity to structural preparation.

A candidate can look convincing in an isolated chain while being inaccessible or incomplete in the relevant complex.

For each candidate, inspect:

1. Assembly and access. Does the site remain plausible with the required partner chains, domains or membrane context?
2. Missing or uncertain atoms. Could unresolved loops or side chains alter the cavity?
3. Non-protein components. Are a metal, cofactor, prosthetic group or retained molecule essential to the proposed site?
4. Source-to-output correspondence. Do chain IDs and residue numbers in the prediction map to the actual receptor used for docking?
5. Chemical context. Is the proposed ligand interaction compatible with the region, rather than merely with an empty geometric volume?

Check how the installed pocket tool treats non-protein components. The original fpocket method, for example, discusses retaining certain cofactors during cavity detection. That historical behavior is not a substitute for checking the current implementation and inspecting the resulting structure. Fpocket preprocessing and algorithm.

Do not delete everything labeled as a heteroatom simply to obtain a cleaner-looking receptor. Record what was retained and why. The separate waters, metals and cofactors guide covers that preparation decision.

A protein-wide confidence summary cannot qualify a particular docking cavity.

For AlphaFold2-derived models, pLDDT describes confidence in local structure. High local confidence in two domains does not necessarily imply confidence in their relative arrangement. PAE helps assess that larger-scale relationship. This distinction matters when a proposed pocket sits between domains rather than within one locally well-supported region. EMBL-EBI on pLDDT, EMBL-EBI on PAE.

The operational consequence is to examine the residues and domain relationships that actually form the candidate site. Confidence is evidence about a model, not a measurement of ligand binding.

Tool configuration matters too. P2Rank 2.5.1 documents an AlphaFold profile that does not depend on B-factor as a feature. Record the profile used rather than treating confidence values stored in a model file as interchangeable with experimental displacement parameters. P2Rank versioned configuration guidance.

Avoid collapsing everything into a single unexplained score. Use a compact record that another researcher can inspect.

Pocket rank alone is not a stable identity across reruns or receptor variants. Preserve the original output and record the residue correspondence used to compare sites.

A hypothetical decision illustrates the distinction: pocket A ranks first but has no functional support and lies near an uncertain domain interface. Pocket B ranks second and overlaps a structurally conserved catalytic region supported by independent evidence. That does not prove B is correct, but it gives a clearer reason to investigate B first. This is a decision example, not an experimental result from P2Rank or fpocket.

A pocket center supplies a location. It does not supply three justified search-space dimensions.

After retaining a site hypothesis, inspect its extent, the intended ligand series and any relevant movement or flexible region. Define and save the box in the same coordinate frame as the receptor. If several sites remain plausible, keep them as separate named hypotheses with separate configurations.

A whole-protein box is not a substitute for this reasoning. It may be appropriate for an explicitly exploratory search, but larger search spaces increase the sampling problem. The AutoDock Vina FAQ discusses the connection between search-space size and search effort.

Do not choose a site solely because it produces a more negative Vina score. Site selection, search effort and pose interpretation are coupled decisions; reporting only the most favorable result hides that dependence.

You cannot calculate cognate redocking RMSD without a suitable known pose of that ligand. Do not manufacture a reference by independently superposing a related compound or selecting the pose that looks most attractive.

Other evidence may still be useful: known actives and appropriate controls, relevant homologous complexes, recurrence under controlled searches, interaction hypotheses, or prospective experiments. Each answers a narrower question. Stable docking into a predicted pocket does not establish that the pocket exists in the relevant biological state.

Defer a large screen when the selected site depends mainly on an uncertain structure, conflicts with the required assembly, or lacks a credible connection to the scientific question. Proceeding with a smaller exploratory comparison can be reasonable if its uncertainty and intended next experiment are explicit.

MolNexus provides visible search-box controls, Vina execution, pose review, exports and local history in a Windows desktop workflow. Pocket prediction and biological site selection remain separate tasks; no built-in P2Rank or fpocket integration is implied.

Once a site is justified, the practical next step is to translate it into a reproducible search-space definition:

Turn a justified site into a controlled Vina search box.