Assessing Structural Prediction Accuracy for Nanobody–Small Molecule Complexes

Abstract

Generative models have transformed structural bioinformatics, enabling antibody and nanobody design against protein epitopes; however, nanobody engineering for small molecule sensing remains largely experimental. In this work, we evaluate seven state-of-the-art structure predictors, AlphaFold3, Chai-1, Boltz-2×, RoseTTAFold3, Protenix, FlowDock and OmegaFold, on nanobody–small molecule complexes. Most predictors accurately reproduced nanobody and CDR geometries but struggled with ligand placement and orientation, although co-folding improved overall accuracy. Contact analysis revealed that CDR1, rather than CDR3, was predominantly involved in ligand binding. Intrinsic confidence scores correlated poorly with experimental accuracy and showed limited power in distinguishing binders from non-binders. Increasing the number of samples and seeds yielded modest gains in accuracy, whereas additional recycles did not. These findings highlight both the strengths and limitations of structure prediction methods for nanobody–small molecule complexes.