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

Menezes, F. M. C. M.; Froehlich, T.; Schofield, L. C.; Cremer, J.; Agirre, J.; Joosten, R. P.; Murshudov, G. N.; Plettenburg, O.; Sattler, M.; Popowicz, G. M.

doi:10.1101/2025.09.24.678379

bioRxiv · 10.1101/2025.09.24.678379

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

Menezes, F. M. C. M.·Froehlich, T.·Schofield, L. C.·Cremer, J.·Agirre, J.·Joosten, R. P.·Murshudov, G. N.·Plettenburg, O.·Sattler, M.·Popowicz, G. M.

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Abstract

Structural biology enabled key advances in biochemistry, drug discovery, and AI-driven three-dimensional structure modelling. Despite its importance, reliable and general computational tools for chemical validation of structural models remain limited, 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 evaluates the chemical nature of protein-ligand binding interfaces. In-Pocket improves model quality, corrects chemically inconsistent structures, and identifies the energetically most favourable ligand-conformers within biomacromolecular complexes. The approach is generally applicable to 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, user-friendly routine chemical validation, In-Pocket offers unique opportunities to improve the integrity of structural databases and impact diverse domains: from molecular biology to rational drug design and application of generative AI.

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Menezes, F. M. C. M., Froehlich, T., Schofield, L. C., Cremer, J., Agirre, J., Joosten, R. P., Murshudov, G. N., Plettenburg, O., Sattler, M., Popowicz, G. M.. 2025-09-27. It is Not Always Crystal Clear: In-Pocket Analysis for Understanding Binding Site Microenvironment Properties. https://doi.org/10.1101/2025.09.24.678379

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