It’s Not Always Crystal Clear: In-Pocket Analysis for Understanding Binding Site Microenvironment Properties

Structural biology enabled key advances in biochemistry, drug discovery, and AI-driven three-dimensional structure modelling. Despite its centrality, no general and reliable computational tools exist to chemically validate structural models, especially when small-molecule ligands are involved. This challenge is exacerbated when experimental resolution is insufficient to define interaction chemistry, leaving critical interpretations ambiguous. We introduce In-Pocket, a quantum mechanical optimisation method biased by experimental data that resolves the chemical nature of protein-ligand binding interfaces. In-Pocket improves model quality, corrects chemically inconsistent structures, and identifies the energetically most favourable conformers within biomacromolecular complexes. Further, it generalises across a wide range of systems, including those containing transition metals. Benchmarking demonstrates that In-Pocket enhances the reliability of legacy and newly solved structures. With automated and user-friendly routine chemical validation, In-Pocket stands to improve the integrity of structural databases and impact diverse domains: from molecular biology to rational drug design and application of generative AI.