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

作者：Jianfang Liu, Ewan K.S. McRae, Meng Zhang, Cody Geary, Ebbe Sloth Andersen, Gang Ren · 发表于：Nature Communications · 年份：2024 · DOI：10.1038/s41467-024-52914-1 · 被引用次数：12 · 研究领域：RNA and protein synthesis mechanisms、Advanced Electron Microscopy Techniques and Applications、RNA modifications and cancer

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.