Non-averaged single-molecule tertiary structures reveal RNA self-folding through individual-particle cryo-electron tomography

Jianfang Liu, Ewan K. S. McRae, Meng Zhang, Cody Geary, Ebbe Sloth Andersen () and Gang Ren ()
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Jianfang Liu: Lawrence Berkeley National Laboratory
Ewan K. S. McRae: Aarhus University
Meng Zhang: Lawrence Berkeley National Laboratory
Cody Geary: Aarhus University
Ebbe Sloth Andersen: Aarhus University
Gang Ren: Lawrence Berkeley National Laboratory

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: Abstract Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and observational scope. Here, we utilize individual-particle cryo-electron tomography (IPET) to examine the post-transcriptional self-folding process of designed RNA origami 6-helix bundle with a clasp helix (6HBC). By avoiding selection, classification, averaging, or chemical fixation and optimizing cryo-ET data acquisition parameters, we reconstruct 120 three-dimensional (3D) density maps from 120 individual particles at an electron dose of no more than 168 e–Å−2, achieving averaged resolutions ranging from 23 to 35 Å, as estimated by Fourier shell correlation (FSC) at 0.5. Each map allows us to identify distinct RNA helices and determine a unique tertiary structure. Statistical analysis of these 120 structures confirms two reported conformations and reveals a range of kinetically trapped, intermediate, and highly compacted states, demonstrating a maturation folding landscape likely driven by helix-helix compaction interactions.

Date: 2024
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DOI: 10.1038/s41467-024-52914-1

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