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| Title | RNA sample optimization for cryo-EM analysis. |
|---|---|
| Journal, issue, pages | Nat Protoc, Vol. 20, Issue 5, Page 1114-1157, Year 2025 |
| Publish date | Nov 15, 2024 |
 Authors | Xingyu Chen / Liu Wang / Jiahao Xie / Jakub S Nowak / Bingnan Luo / Chong Zhang / Guowen Jia / Jian Zou / Dingming Huang / Sebastian Glatt / Yang Yang / Zhaoming Su /     |
| PubMed Abstract | RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine ...RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations. |
 External links | Nat Protoc / PubMed:39548288 |
| Methods | EM (single particle) |
| Resolution | 6.59 Å |
| Structure data |  EMDB-37072: Cryo-EM structure of dimeric ATP-TTR-3 |
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