制备用于高分辨率冷冻电镜成像的冷冻水合蛋白质-核酸组装体。

Preparing Frozen-Hydrated Protein-Nucleic Acid Assemblies for High-Resolution Cryo-EM Imaging.

作者信息

Goswami Panchali, Locke Julia, Costa Alessandro

机构信息

Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK.

出版信息

Methods Mol Biol. 2018;1814:287-296. doi: 10.1007/978-1-4939-8591-3_17.

DOI:10.1007/978-1-4939-8591-3_17

PMID:29956239

Abstract

High-resolution image acquisition and structure determination by cryo-electron microscopy is becoming increasingly streamlined. Preparing electron-microscopy grids of suitable quality remains, however, a critical bottleneck. Strategies to achieve particle monodispersity, optimal sample concentration and suitable ice thickness can vary from specimen to specimen. In this book chapter we describe our protocols for negative-stain grid and cryo-grid preparation, which we apply to studying protein-nucleic acid complexes.

摘要

通过冷冻电子显微镜进行高分辨率图像采集和结构测定正变得越来越简化。然而，制备质量合适的电子显微镜网格仍然是一个关键瓶颈。实现颗粒单分散性、最佳样品浓度和合适冰厚度的策略可能因样品而异。在本章中，我们描述了用于负染网格和冷冻网格制备的方案，这些方案我们用于研究蛋白质 - 核酸复合物。