RNA molecules perform numerous important biological functions including sensing small molecules, regulating gene expression, and catalyzing reactions in all living systems. The three-dimensional structures of these RNA molecules dictate their functions, but remain challenging to determine experimentally. Here we describe a hybrid method for RNA structure determination integrating cryo-EM and mutate-and-map-seq (M2-seq) secondary structure determination with Rosetta computational modeling. Tests of this method both in blind challenges and with simulated data suggest that the resulting 3D models are highly accurate and that model accuracy can be reliably predicted. Using this method, we rapidly determined 14 novel RNA structures including ribozymes, riboswitches, and computationally designed RNA molecules. The sub-nanometer resolution structural models provide a wealth of functional insights and demonstrate that this hybrid pipeline enables rapid, unbiased RNA structure determination.

Rapid RNA structure determination through cryo-EM, high-throughput biochemistry, and computational modeling

2019 Fall National Meeting

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