Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Friday Speaker Abstracts

ACCURATE PROTEIN FITTING INTO CRYO-EM MAPS USING MULTIPLE CONFORMERS GENERATED BY ALPHAFOLD2 Tatiana Shugaeva 1 ; Nandan Haloi 1 ; Rebecca J Howard 1,2 ; Erik Lindahl 1,2 ; 1 KTH Royal Institute of Technology, Department of Applied Physics, Stockholm, Sweden 2 Stockholm University, Department of Biochemistry and Biophysics, Stockholm, Sweden Recent advances in cryo-electron microscopy have enabled near-atomic resolution structures of challenging protein targets. Refining an initial atomistic model into a target density map is one of the key reconstruction steps, where molecular dynamics simulations can be used to achieve a good balance between physical plausibility and map fitting scores. However, proteins with multiple functional states often remain a challenge for simulation-based refinement methods, in particular when map resolution is not high enough to build de-novo models. Here, we introduce a new refinement approach in which 1) numerous initial models (instead of a single one) are generated by stochastic subsampling the multiple sequence alignments (MSA) space in AlphaFold2, 2) resulting models are subjected to structure-based clustering, and 3) multiple density-guided molecular dynamics simulations are performed from the centroid structures. Our method shows better fitting accuracy compared to single starting point scenarios. This improvement is demonstrated for the epsilon subunit of ATP-synthase, CGRPR, LAT1, and ASCT2. Each of these proteins undergoes significant conformational changes. The results suggest that simulation-based refinement combined with AlphaFold2-MSA-subsampling might both make it possible to model transitions between states and build models of states with smaller populations in cryo-EM data. This could be especially beneficial for drug design applications because it might help in the identification of compounds that selectively stabilize specific states.

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