Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures

Abstract

The discovery and design of biologically important RNA molecules is outpacing threedimensional structural characterization. Here, we demonstrate that cryo-EM can routinely resolve maps of RNA-only systems and that these maps enable sub-nanometer resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in eleven previously unknown 119-to 338nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length V. cholerae and F. nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies, and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules, but does not Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

“…The map also showed non-helical crevices and holes suggestive of potential small molecule binding pockets ( Fig. 2c), as previously observed and validated in cryo-EM maps of SAM-IV riboswitch aptamers with and without small molecule ligands 7,37 .…”

“…2e, Extended Data Fig. 7 and Extended Data Movie 1), corresponding to a 5.9-Å estimated accuracy, based on a linear relationship of precision and accuracy discovered previously 7 , with the largest uncertainty in the 5′ end and at Stem 2 (estimated accuracy of >10 Å; Extended Data Fig. 8).…”

“…Cryo-EM structure determination of RNA-only systems or macromolecules with molecular weights under 50 kDa has been accomplished only recently 7,[35][36][37][38] , and it was unclear whether cryo-EM might be applicable to an RNA as small as the SARS-CoV-2 FSE (28 kDa, 88 nucleotides; Fig. 1a).…”

“…3a). For the tag, we chose the rationally designed ATP-TTR 3 RNA 47 based on its known amenability to cryo-EM imaging 7 and on modeling suggesting that its insertion into the FSE would not perturb either RNAs' secondary structure, a prediction verified through chemical mapping (Extended Data Fig. 10).…”

“…The RNA samples were folded prior to plunge freezing as previously described 7 movie stacks for FSE and 12,380 movie stacks for FSE-ATP-TTR3 were collected.…”