Biophysical Society Thematic Meeting | Trieste 2024

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

ALL ATOM MOLECULAR DYNAMICS SIMULATIONS ENABLE ENSEMBLE REFINEMENT OF FLEXIBLE AND MISMODELLED CRYO-EM DERIVED RNA STRUCTURES Elisa Posani 1 ; Pavel Janoš 2 ; Massimiliano Bonomi 3 ; Alessandra Magistrato 2 ; Giovanni Bussi 1 ; 1 SISSA, Trieste, Italy 2 CNR-IOM, Trieste, Italy 3 CNRS - Institut Pasteur, Paris, France RNA molecules span a great variety of biological functions, from genetic information storage to catalysis. This can be achieved thanks to the highly heterogeneous conformational ensembles that these molecules can adopt [1]. With the advent of the single particle cryogenic electron microscopy (cryo-EM) technique, it is possible to access large and highly flexible RNA macromolecules at near atomic-level resolution, thus allowing to fully assess their structural and functional features. Still, given that standard refinement tools assume that all the collected images are associated to a single structure, the most mobile regions are very challenging to solve. We tackle these problems by integration of molecular dynamics simulations and experimental density maps, using as a test case a group IIB intron ribozyme [2]. We show that a refinement done assuming a single structure leads to group II intron structural models with nucleobase base pairs that are either non-properly paired or in disagreement with the experimental potential density map. Interestingly, incorrect base pairing is also present in the deposited structure [2]. We solve this problem by using metainference-based [3] ensemble refinement, along with ad hoc restraints to enforce the correct base pairing. The use of an ensemble refinement approach enables the natural RNA dynamics to be reconstructed, preserving the proper interactions.[1] J.A. Doudna, T.R. Cech, Nature, 418: 222-228 (2002).[2] D.B. Haak et al., Cell, 178: 612-623 (2019).[3] M. Bonomi et al., Science Advances, 2: 3 (2016).

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