Scaffolds with optimized quaternary symmetry for de novo cryoEM structure determination of small RNAs
Christopher P. Jones, Adrian R. Ferré-D’Amaré
TL;DR
Researchers developed symmetric RNA scaffolds to determine high-resolution structures of small RNAs using cryoEM, enabling detailed analysis of their architecture.
Contribution
The novel use of symmetric RNA scaffolds allows cryoEM structure determination of small RNAs previously too tiny for this method.
Findings
C2 and D2-symmetric scaffolds enabled cryoEM structures of small RNAs at resolutions beyond 3 Å.
The method was applied to tRNAAsp, Mango-III, and new aptamers, revealing molecular details of RNA-ligand interactions.
The scaffolds provide a general tool for studying compact RNA folds at the atomic level.
Abstract
Structured RNAs play many roles in cells and emerging biotechnology. While large RNAs and ribonucleoprotein complexes often benefit from high-resolution structural analysis through cryogenic-sample electron microscopy (cryoEM), single-domain RNAs, particularly those smaller than ~100 nt (33 kDa), have proven challenging. Here we address this methodological gap by engineering two- and fourfold symmetric scaffolds that enable de novo structure solution of covalently attached RNA guests to beyond 3 Å overall resolution for the best resolved guests. We apply C2 and D2-symmetric scaffolds to post-transcriptionally unmodified tRNAAsp, the fluorogenic aptamer Mango-III, and previously uncharacterized quinine- and 8-oxoguanine-binding aptamers. Experimental Coulomb potential maps with quality sufficient for small-molecule ligand, cation and water molecule placement reveal the molecular basis…
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Taxonomy
TopicsAdvanced Electron Microscopy Techniques and Applications · RNA modifications and cancer · RNA and protein synthesis mechanisms