Pose validation

Version Compatibility

Reference examples tested with: PoseBusters 0.6+, RDKit 2024.09+, pandas 2.2+, posecheck 0.5+ (optional).

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Pose Validation

Test docked or AI-generated protein-ligand poses for physical plausibility. PoseBusters (Buttenschoen et al. 2024) provides geometric, chemical, and energetic checks that flag implausible poses, including non-planar aromatic rings, van der Waals clashes, broken bonds, altered stereochemistry, and unfavorable internal energies. On the Astex Diverse Set, DiffDock achieved 72% RMSD success but only 47% combined RMSD-and-PB-valid success; the size of this gap is dataset- and method-dependent. PB-valid status complements RMSD for downstream SAR, FEP setup, or generative-model training.

For docking, see chemoinformatics/virtual-screening. For ML docking specifically, see chemoinformatics/ml-docking-rescoring.

PoseBusters Test Suite

PoseBusters runs ~20 individual checks grouped into:

The thresholds below are the benchmark criteria reported by Buttenschoen et al. (2024). Installed PoseBusters defaults may differ by version and configuration, so record the package version and resolved configuration.

Check group	What it tests	2024 benchmark criterion
Sanity	Ligand chemical sanity	RDKit sanitization passes
Bond lengths	Bond lengths within reference	0.75–1.25 times RDKit distance-geometry bounds
Bond angles	1–3 distances within reference	0.75–1.25 times RDKit distance-geometry bounds
Internal steric	No intra-ligand clash	Pair distance > 0.70 times the RDKit lower bound
Aromatic ring planarity	Aromatic rings planar	Maximum deviation from fitted plane <= 0.25 Å
Double-bond stereo	Z/E preserved	Match input SMILES
Internal energy	Energy relative to generated conformers	UFF energy ratio <= 100 versus the mean of 50 generated, relaxed conformers
Volume overlap	vdW overlap with protein	< 7.5% of ligand vdW volume
Minimum distance	No severe protein-ligand clash	Distance >= 0.75 times the sum of vdW radii
Chirality	R/S preserved from input	Match input SMILES

A pose passing ALL tests is "PB-valid". Combined PB-valid + RMSD <= 2 Å is the modern criterion.

When to Apply PoseBusters

Workflow	PoseBusters use	Action
Self-docking (validating method)	Required	Compare PB-valid + RMSD <= 2A
Cross-docking	Required	PB-valid + RMSD <= 2A; account for protein flexibility
Virtual screening top hits	Required	Filter to PB-valid before MM/GBSA / FEP
AI docking (DiffDock, etc.)	Required for a fair benchmark	Report the dataset-specific PB-valid and combined success rates
Generated ligand poses	Recommended	Measure chemical and geometric validity rather than assuming it
Boltz-2 / AlphaFold3 ligand poses	Recommended	Benchmark validity on the relevant complexes; do not infer a failure frequency from DiffDock
Production FEP setup	Required	Inspect pose validity and ligand strain before system preparation

PoseBusters Usage

from posebusters import PoseBusters

bust = PoseBusters(config='redock')

results = bust.bust(
    mol_pred='predicted.sdf',
    mol_true='reference.sdf',
    mol_cond='receptor.pdb',
)

Common configurations and their included checks are:

Config	Includes	When to use
redock	All checks + RMSD vs reference + protein vdW overlap	Self-docking benchmarks, retrospective validation
dock	All checks except RMSD reference	Blind docking, prospective virtual screening
mol	Intra-ligand only (sanity, bonds, angles, rings, stereo, energy)	Conformer QC; no protein context

PoseBusters also ships additional and faster configurations in some releases. Treat the table as a workflow guide, not an exhaustive registry, and inspect the configurations available in the installed version.

Output: a DataFrame with one row per pose, metadata columns, and boolean pass/fail columns for the checks enabled by the selected configuration. Reference-dependent fields such as RMSD and the exact check-column names vary by configuration and version; inspect results.columns rather than relying on a fixed exhaustive list.

Python Library API

Goal: Programmatically validate a docked-pose SDF against a receptor PDB and produce a PB-valid filter.

Approach: Instantiate PoseBusters(config='dock'), call bust() on the SDF + PDB pair, and AND-aggregate all boolean check columns into a single pb_valid flag.

from posebusters import PoseBusters
import pandas as pd

bust = PoseBusters(config='dock')

results = bust.bust(
    mol_pred='/path/to/docked_poses.sdf',
    mol_cond='/path/to/receptor.pdb',
)

check_cols = [
    col for col in results.select_dtypes(include='bool').columns
    if not col.lower().startswith('rmsd')
]
results['pb_valid'] = results[check_cols].all(axis=1)
valid = results[results['pb_valid']]
print(f'{len(valid)} / {len(results)} poses are PB-valid')

Strain Energy Quantification

Beyond binary PB-valid, quantitative strain energy distinguishes "marginal" from "egregious" poses.

Goal: Quantify how far each docked pose is from its lowest-energy free conformer in MMFF94 energy units.

Approach: Generate a reference conformer ensemble (ETKDGv3 + MMFF94), make the docked and reference molecules chemically consistent by adding explicit hydrogens to both, relax only the added docked-pose hydrogens while fixing all heavy atoms, take the lowest sampled reference energy as baseline, and report docked_energy - min_ref_energy as a relative strain diagnostic. This is not a rigorous solution-phase conformational free energy.

from rdkit import Chem
from rdkit.Chem import AllChem

def ligand_strain(docked_sdf, n_ref=20):
    suppl = Chem.SDMolSupplier(docked_sdf, removeHs=False)
    strains = []
    for docked in suppl:
        if docked is None:
            continue

        smi = Chem.MolToSmiles(docked)
        ref = Chem.MolFromSmiles(smi)
        if ref is None:
            strains.append({'strain': None, 'note': 'reference_parse_failed'})
            continue
        ref = Chem.AddHs(ref)
        props_ref = AllChem.MMFFGetMoleculeProperties(ref)
        if props_ref is None:
            strains.append({'strain': None, 'note': 'no_reference_mmff_parameters'})
            continue
        conf_ids = list(AllChem.EmbedMultipleConfs(
            ref, numConfs=n_ref, params=AllChem.ETKDGv3()
        ))
        if not conf_ids:
            strains.append({'strain': None, 'note': 'reference_embedding_failed'})
            continue
        AllChem.MMFFOptimizeMoleculeConfs(ref)

        ref_energies = []
        for c in conf_ids:
            ff = AllChem.MMFFGetMoleculeForceField(
                ref, props_ref, confId=c
            )
            if ff is not None:
                ref_energies.append(ff.CalcEnergy())
        if not ref_energies:
            strains.append({'strain': None, 'note': 'reference_force_field_failed'})
            continue
        min_ref = min(ref_energies)

        # MMFF energies are comparable only for the same explicit atom system.
        # Add any missing H coordinates, then relax H atoms while preserving the
        # docked heavy-atom pose.
        docked_h = Chem.AddHs(Chem.Mol(docked), addCoords=True)
        if docked_h.GetNumAtoms() != ref.GetNumAtoms():
            strains.append({'strain': None, 'note': 'atom_system_mismatch'})
            continue
        props_docked = AllChem.MMFFGetMoleculeProperties(docked_h)
        docked_ff = AllChem.MMFFGetMoleculeForceField(
            docked_h, props_docked
        ) if props_docked is not None else None
        if docked_ff is not None:
            for atom in docked_h.GetAtoms():
                if atom.GetAtomicNum() != 1:
                    docked_ff.AddFixedPoint(atom.GetIdx())
            docked_ff.Minimize(maxIts=200)
        docked_e = docked_ff.CalcEnergy() if docked_ff else None

        strains.append({
            'min_ref_energy': min_ref,
            'docked_energy': docked_e,
            'strain': docked_e - min_ref if docked_e is not None else None,
            'note': 'ok' if docked_e is not None else 'docked_force_field_failed',
        })
    return strains

Interpret relative MMFF strain in the context of ligand chemistry, conformer-sampling coverage, and force-field support. Boström et al. (1998) found a conformational energy penalty of no more than 3 kcal/mol for about 70% of 33 protein-bound ligands; that result does not establish a universal acceptance cutoff. Treat unusually high values as a prompt for inspection or use a project-defined threshold validated for the series.

vdW Overlap with Protein

The 2024 benchmark criterion limits protein-ligand overlap to 7.5% of the ligand vdW volume, using protein radii scaled by 0.8. PoseBusters' bust(...) computes this check; do not substitute an unvalidated pairwise-distance sketch for its volume calculation.

Aromatic Ring Planarity

import numpy as np

def aromatic_planarity(mol):
    deviations = []
    for ring in mol.GetRingInfo().AtomRings():
        ring_atoms = [mol.GetAtomWithIdx(i) for i in ring]
        if not all(a.GetIsAromatic() for a in ring_atoms):
            continue
        coords = np.array([mol.GetConformer().GetAtomPosition(i)
                          for i in ring])
        centroid = coords.mean(axis=0)
        centered = coords - centroid
        _, s, vh = np.linalg.svd(centered)
        normal = vh[-1]
        deviation = np.abs(centered @ normal).max()
        deviations.append(deviation)
    return max(deviations) if deviations else 0

Aromatic ring deviation > 0.25 Å is implausible; flag.

Model-Specific Failure Diagnosis

Do not assign a mechanism from the model name or a failed PoseBusters column alone. For DiffDock-L, EquiBind, TANKBind, Boltz, AlphaFold3, or another pose generator, report the observed failed checks on the evaluated dataset, inspect the structures, and compare against the method's documented constraints. A chirality, planarity, bond-geometry, or clash failure may justify filtering or a validated constrained-relaxation protocol, but relaxation must be checked for displacement of the binding mode.

High strain after Vina docking

Trigger: Highly constrained pocket; flexible ligand.

Symptom: Relative strain is an outlier for the chemical series even though the pose passes the enabled geometric checks.

Fix: Inspect conformer-sampling coverage and force-field support. Compare additional docking or constrained-relaxation settings under a project-validated protocol rather than applying a universal strain or exhaustiveness cutoff.

Reconciliation: PoseBusters vs RMSD

RMSD <= 2A	PB-valid	Action
Yes	Yes	Physically plausible and close to the reference; still validate suitability for the downstream task
Yes	No	Close to the reference but fails an enabled plausibility check; inspect the failure and any validated relaxation
No	Yes	Physically plausible but different from the reference; investigate alignment, protein state, and alternative binding modes
No	No	Different from the reference and fails an enabled plausibility check; inspect both causes before deciding whether to reject

On the Astex Diverse Set reported by Buttenschoen et al. (2024), DiffDock's top-pose success fell from 72% by RMSD <= 2 Å alone to 47% when PB-validity was also required: a 25-percentage-point gap. Do not generalize that result to a fixed failure rate on other datasets.

Integration into VS Pipeline

import pandas as pd
from posebusters import PoseBusters

def pose_qc_pipeline(docked_sdfs, receptor_pdb):
    bust = PoseBusters(config='dock')
    all_results = []
    for sdf in docked_sdfs:
        r = bust.bust(mol_pred=sdf, mol_cond=receptor_pdb)
        check_cols = [
            col for col in r.select_dtypes(include='bool').columns
            if not col.lower().startswith('rmsd')
        ]
        r['pb_valid'] = r[check_cols].all(axis=1)
        r['source'] = sdf
        all_results.append(r)
    df = pd.concat(all_results)

    df['rank'] = df.groupby('source')['pb_valid'].cumsum()
    valid_top = df[df['pb_valid']].groupby('source').head(1)
    return valid_top

Common Errors

Symptom	Cause	Fix
Rows or expected checks are missing	Input loading failed or the selected configuration omits those checks	Inspect the returned DataFrame, loading-status columns, input format, and installed configuration
RMSD not computed	No reference provided	Pass mol_true parameter
All checks pass for invalid pose	Wrong receptor file format	Use PDB with hydrogens; PDBQT may not work
vdW overlap false positive on covalent	Covalent bond counted as clash	Use covalent docking-specific validation
Strain calculation slow	Too many reference conformers	Reduce n_ref to 5-10
PoseBusters config error	Wrong or version-incompatible config name	Inspect the installed configuration registry; redock, dock, and mol are common configurations
posecheck unavailable	Different tool, similar purpose	pip install posecheck for alternative

References

• Buttenschoen M, Morris GM, Deane CM. "PoseBusters: AI-based docking methods fail to generate physically valid poses or generalise to novel sequences." Chem. Sci. 15:3130–3139 (2024). DOI: 10.1039/D3SC04185A.
• Boström J, Norrby PO, Liljefors T. "Conformational energy penalties of protein-bound ligands." J. Comput.-Aided Mol. Des. 12:383–396 (1998). DOI: 10.1023/A:1008007507641.
• Corso G et al. "DiffDock: Diffusion Steps, Twists, and Turns for Molecular Docking." ICLR (2023). OpenReview: https://openreview.net/forum?id=kKF8_K-mBbS.
• Stärk H et al. "EquiBind: Geometric Deep Learning for Drug Binding Structure Prediction." PMLR 162:20503–20521 (2022). https://proceedings.mlr.press/v162/stark22b.html.
• Lu W et al. "TankBind: Trigonometry-Aware Neural NetworKs for Drug-Protein Binding Structure Prediction." NeurIPS 35 (2022). Official repository: https://github.com/luwei0917/TankBind.
• Abramson J et al. "Accurate structure prediction of biomolecular interactions with AlphaFold 3." Nature 630:493–500 (2024). DOI: 10.1038/s41586-024-07487-w.
• Boltz official repository and documentation: https://github.com/jwohlwend/boltz.
• PoseBusters documentation, Python API: https://posebusters.readthedocs.io/en/latest/api.html.

Related Skills

• chemoinformatics/virtual-screening - Source of poses to validate
• chemoinformatics/ml-docking-rescoring - DiffDock, EquiBind, TANKBind validation
• chemoinformatics/molecular-io - SDF format handling
• chemoinformatics/conformer-generation - Generate reference conformer ensemble for strain
• chemoinformatics/free-energy-calculations - PoseBusters-valid poses for FEP input
• chemoinformatics/covalent-design - Covalent pose validation