Scaffold-enabled high-resolution cryo-EM structure determination of RNA

Daniel B. Haack, Boris Rudolfs, Shouhong Jin, Alexandra Khitun, Kevin M. Weeks and Navtej Toor ()
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Daniel B. Haack: University of California
Boris Rudolfs: University of California
Shouhong Jin: University of North Carolina
Alexandra Khitun: University of North Carolina
Kevin M. Weeks: University of North Carolina
Navtej Toor: University of California

Nature Communications, 2025, vol. 16, issue 1, 1-17

Abstract: Abstract Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

Date: 2025
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DOI: 10.1038/s41467-024-55699-5

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