MOE Key Laboratory for Cellular Dynamics and Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Department of Bioengineering, James H. Clark Center, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2023;2568:179-192. doi: 10.1007/978-1-0716-2687-0_12.
The rapid development of cryogenic electron microscopy (cryo-EM) enables the structure determination of macromolecules without the need for crystallization. Protein, protein-lipid, and protein-nucleic acid complexes can now be routinely resolved by cryo-EM single-particle analysis (SPA) to near-atomic or atomic resolution. Here we describe the structure determination of pure RNAs by SPA, from cryo-specimen preparation to data collection and 3D reconstruction. This protocol is useful to yield many cryo-EM structures of RNA, here exemplified by the Tetrahymena L-21 ScaI ribozyme at 3.1-Å resolution.
低温电子显微镜(cryo-EM)的快速发展使得在无需结晶的情况下也能够确定生物大分子的结构。现在,通过 cryo-EM 单颗粒分析(SPA)可以常规解析蛋白质、蛋白脂质体和蛋白核酸复合物,达到近原子或原子分辨率。在这里,我们描述了通过 SPA 来确定纯 RNA 的结构,从 cryo-样品制备到数据收集和 3D 重构。该方案有助于获得许多 RNA 的 cryo-EM 结构,这里以 3.1-Å分辨率的 Tetrahymena L-21 ScaI 核酶为例。