Practical Outcomes From CASP16 for Users in Need of Biomolecular Structure Prediction

ABSTRACT The 16th Critical Assessment of Structure Prediction benchmarked advancements in biomolecular modeling, particularly in the context of AlphaFold 2 and 3 systems. Protein monomer and domain prediction is largely solved, with barely any space for further improvements at the backbone level except for very specific details, irregular secondary structures, and mutational effects that remain challenging to predict. For protein assemblies, AF‐based methods, especially when expertly guided or enhanced by servers like those from the Yang, Zheng/Zhang, and Cheng lab, show progress, though complex topologies and in particular antibody–antigen interactions are still difficult. Notably, a priori knowledge of stoichiometry significantly aids assembly prediction. Protein‐ligand co‐folding with AF3 demonstrated strong potential for pose prediction, outperforming many participants and some dedicated docking tools in baseline tests, but several caveats hold as discussed. Ligand affinity prediction is totally unreliable. Nucleic acid structure prediction lags considerably, heavily relying on 3D templates and expert human intervention, even AF3 showing substantial limitations. Overall, on all fronts, AF3's modeling capabilities are at or close to the state of the art; additionally, it shows slight improvements over AF2 and more detailed confidence metrics than it. We guide users on tool selection, realistic accuracy expectations, and persistent challenges, emphasizing the critical role of confidence metrics in interpreting AI‐generated models.