RNA sample optimization for cryo-EM analysis.

Autor: Chen X; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China., Wang L; The State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, National Center for Stomatology, Department of Cardiology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China., Xie J; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China., Nowak JS; Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland., Luo B; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China.; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA., Zhang C; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China., Jia G; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China., Zou J; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China., Huang D; The State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, National Center for Stomatology, Department of Cardiology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China., Glatt S; Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.; Department for Biological Sciences and Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria., Yang Y; Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China., Su Z; The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China. zsu@wchscu.cn.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2024 Nov 15. Date of Electronic Publication: 2024 Nov 15.
DOI: 10.1038/s41596-024-01072-1
Abstrakt: 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.
Competing Interests: Competing interests The authors declare no competing interests.
(© 2024. Springer Nature Limited.)
Databáze: MEDLINE
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