Validates docked / generated protein-ligand poses using PoseBusters physical-validity tests, strain energy quantification, geometric checks (planarity, vdW overlap, bond/angle distortion), and pose-energy reasonableness. Use when QC-ing docking results, comparing classical vs ML docking outputs, or filtering pose lists before SAR analysis.
Validates docked / generated protein-ligand poses using PoseBusters physical-validity tests, strain energy quantification, geometric checks (planarity, vdW overlap, bond/angle distortion), and pose-energy reasonableness. Use when QC-ing docking results, comparing classical vs ML docking outputs, or filtering pose lists before SAR analysis.
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 . For ML docking specifically, see .
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
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
ifnot 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
defligand_strain(docked_sdf, n_ref=20):
suppl = Chem.SDMolSupplier(docked_sdf, removeHs=False)
strains = []
for docked in suppl:
if docked isNone:
continue
smi = Chem.MolToSmiles(docked)
ref = Chem.MolFromSmiles(smi)
if ref isNone:
strains.append({'strain': None, 'note': 'reference_parse_failed'})
continue
ref = Chem.AddHs(ref)
props_ref = AllChem.MMFFGetMoleculeProperties(ref)
if props_ref isNone:
strains.append({'strain': None, 'note': 'no_reference_mmff_parameters'})
continue
conf_ids = list(AllChem.EmbedMultipleConfs(
ref, numConfs=n_ref, params=AllChem.ETKDGv3()
))
ifnot 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 isnotNone:
ref_energies.append(ff.CalcEnergy())
ifnot 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 isnotNoneelseNoneif docked_ff isnotNone:
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 elseNone
strains.append({
'min_ref_energy': min_ref,
'docked_energy': docked_e,
'strain': docked_e - min_ref if docked_e isnotNoneelseNone,
'note': 'ok'if docked_e isnotNoneelse'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
defaromatic_planarity(mol):
deviations = []
for ring in mol.GetRingInfo().AtomRings():
ring_atoms = [mol.GetAtomWithIdx(i) for i in ring]
ifnotall(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)
returnmax(deviations) if deviations else0
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.
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
defpose_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
ifnot 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.
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.
