Hyun Jaekyung, Hsieh Louise Tzung-Harn, Ayala Rafael, Chang Wen, Wolf Matthias
School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi, Republic of Korea.
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
Methods Mol Biol. 2025;2860:191-218. doi: 10.1007/978-1-0716-4160-6_13.
Poxviruses are double-stranded DNA viruses that represent the largest known highly pathogenic viruses infecting humans. They undergo dramatic morphological changes during their maturation process, resulting in structural differences between each virion, and their surface is decorated with more than a dozen randomly distributed surface proteins that facilitate viral entry. These are the main reasons poxviruses have eluded high-resolution structure determination. Over the last three decades, cryo-EM has developed into a mature technology that can increasingly overcome such problems of structural heterogeneity through advances in microscope technology and image processing algorithms. Here, we discuss the essential current modalities in cryo-EM, which promise to solve the structure of poxviruses in parts and as entire virions at near-atomic resolution. With a focus on cryo modalities, we provide an overview of methods, including volume microscopy by plasma ion beam milling, focused ion beam lamella preparation, subtomogram averaging, and single particle averaging. Protocols for poxvirus propagation, purification, and imaging by cryo-EM are presented. This chapter is aimed at experts and nonexpert researchers to help facilitate entry into the structural biology of this critical field in virology.
痘病毒是双链DNA病毒，是已知感染人类的最大的高致病性病毒。它们在成熟过程中会发生显著的形态变化，导致每个病毒粒子之间存在结构差异，并且其表面装饰有十几种随机分布的表面蛋白，这些蛋白有助于病毒进入。这些是痘病毒难以进行高分辨率结构测定的主要原因。在过去三十年中，冷冻电子显微镜已发展成为一项成熟的技术，通过显微镜技术和图像处理算法的进步，能够越来越多地克服结构异质性等问题。在此，我们讨论了当前冷冻电子显微镜中的基本模式，这些模式有望以接近原子分辨率解析痘病毒部分结构及整个病毒粒子的结构。我们重点关注冷冻模式，概述了相关方法，包括等离子体离子束铣削的体积显微镜、聚焦离子束薄片制备、亚断层平均和单颗粒平均。还介绍了痘病毒增殖、纯化及冷冻电子显微镜成像的方案。本章旨在帮助专家和非专家研究人员进入病毒学这一关键领域的结构生物学研究。
