Improving AlphaFold2 and 3-based protein complex structure prediction with MULTICOM4 in CASP16.

With AlphaFold achieving high-accuracy tertiary structure prediction for most single-chain proteins (monomers), the next major challenge in protein structure prediction is accurately modeling multi-chain protein complexes (multimers). We developed MULTICOM4, the latest version of the MULTICOM system, to improve protein complex structure prediction by integrating transformer-based AlphaFold2, diffusion model-based AlphaFold3, and our in-house techniques. These include protein complex stoichiometry prediction, diverse multiple sequence alignment (MSA) generation leveraging both sequence and structure comparison, modeling exception handling, and deep learning-based model quality assessment. MULTICOM4 was blindly evaluated in the 16th community-wide Critical Assessment of Techniques for Protein Structure Prediction (CASP16) in 2024. In Phase 0 of CASP16, where stoichiometry information was unavailable, MULTICOM predictors performed best, with MULTICOM_human achieving a TM-score of 0.752 and a DockQ score of 0.584 for top-ranked predictions on average. In Phase 1 of CASP16, with stoichiometry information provided, MULTICOM_human remained among the top predictors, attaining a TM-score of 0.797 and a DockQ score of 0.558 on average. The CASP16 results demonstrate that integrating complementary AlphaFold2 and 3 with enhanced MSA inputs, comprehensive model ranking, exception handling, and accurate stoichiometry prediction can effectively improve protein complex structure prediction.