Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM Wednesday Speaker Abstracts

CHARACTERISING THE CONFORMATIONAL LANDSCAPE OF THE HEXAMERIC VCP COMPLEX FROM 2D CRYO-EM IMAGES USING MOLECULAR DYNAMICS SIMULATION Remi Vuillemot 1,2 ; Sepideh Valimehr 1,3 ; Mohsen Kazemi 1,3 ; Slavica Jonic 2 ; Isabelle Rouiller 1,3 ; 1 The University of Melbourne, Biochemistry & Pharmacology, Bio21 Institute, Melbourne, Australia 2 Sorbonne Université, IMPMC-UMR 7590 CNRS, Muséum National d'Histoire Naturelle, Paris, France 3 Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Parkville, Australia The different conformations adopted by protein complexes in solution are captured in cryo-EM data. However, retrieving this information remains challenging. Classical image analysis methods rely on averaging, which is inadequate for obtaining detailed information on the flexible regions of proteins. The calculated EM maps have poorly defined or invisible densities for regions of complexes that undergo continuous conformational changes. To analyse the conformational dynamics of proteins from cryo-EM data, we have developed a method that combines Normal Mode Analysis and Molecular Dynamics simulation to deform/fit 3D structures to match the 2D cryo-EM images data. Using this method, called Molecular Dynamics for Single Particle Analysis of Continuous Conformational hEterogeneity or MDSPACE [1], we analyzed the conformational variability in the hexameric AAA + ATPase p97/VCP, a protein complex with a six-fold rotational symmetric core surrounded by six flexible N-domains [2]. This approach enabled us to detect and characterize the swaying moving of a ~30KDa domain, the N-domain of VCP, by up to 60° around a central position [3]. It also enabled us to analyze the cooperativity of the six N-domains within the VCP hexamer. Moreover, cluster analysis of the 3D models fitted to the 2D images revealed the presence of an unexpected conformation adopted by very few particles in the data set (approximately 2%). This study demonstrates the application of MDSPACE in analysing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyse due to the alignment errors caused by their partial symmetry. References: 1. Vuillemot, R., et al., J Mol Biol, 2023: p. 167951; 2. Valimehr, S., et al., Biomolecules, 2023. 13(5); 3. Valimehr, S., et al., Int J Mol Sci, 2024. 25(6).

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