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

• Published in: Nature communications 2024-10, Vol.15 (1), p.9084-18, Article 9084
• Main Authors: Liu, Jianfang, McRae, Ewan K. S., Zhang, Meng, Geary, Cody, Andersen, Ebbe Sloth, Ren, Gang
• Format: Article
• Language: English
• Subjects: 101/28; 147/143; 631/1647/245/2160; 631/45/500; 631/535/1258/1260; 631/57/2265; Cryoelectron Microscopy - methods; Data acquisition; Electron Microscope Tomography - methods; Folding; Helices; Humanities and Social Sciences; Maturation; Models, Molecular; multidisciplinary; Nucleic Acid Conformation; Post-transcription; Protein structure; Ribonucleic acid; RNA; RNA - chemistry; RNA Folding; Science; Science (multidisciplinary); Statistical analysis; Tertiary structure; Tomography
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-52914-1

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. Using cryogenic electron tomography (cryo-ET) and individual-particle cryo-electron tomography (IPET), non-averaged ternary structures of individual RNA origami 6HBC particles were determined, allowing observation of structural diversity and self-folding dynamics during the maturation process after transcription.