Bacterial Secretion Systems - Methods and Protocols
Bacterial Secretion Systems - Methods and Protocols
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细菌分泌系统 - 方法和方案
DOI：
10.1007/978-1-0716-3445-5_27
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Ignatiou A
中科院分区：
文献类型：
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作者：
Ignatiou A
使用单粒子低温电子显微镜（cryo-EM）对生物复合物进行结构研究是当今结构生物学中的一种成熟技术，并且已经与X射线晶体学竞争。电子显微镜图像的数字配准系统和算法的快速和有效的处理记录的图像及其后续分析的发展，促进了近原子分辨率的结构的确定。EM的最新进展使得能够以1.4-3 kDa的分辨率确定蛋白质复合物结构，其尺寸范围非常宽（从~100 kDa到数百MDa）（Bartesaghi等人，Science 348（6239）：1147-1151，2015; Herzik等人，Nat Commun 10：1032，2019; Wu等人，J Struct Biol X 4：100020，2020; Zhang等人，Nat Commun 10：5511，2019; Zhang等人，Cell Res 30（12）：1136-1139，2020; Yip等人，Nature 587（7832）：157-161，2020; https：//www.ebi.ac.uk/emdb/acetics/emdb_resolution_year））。在2022年，近1200个存储到EMDB数据库的结构的分辨率超过3 μ m（https：//www.ebi.ac.uk/emdb/applications/emdb_resolution_year）。迄今为止，已经实现了脱铁铁蛋白的最高分辨率，其包含高点群对称性的同源寡聚体（O 432），并且具有刚性组织和高稳定性（Zhang et al.，Cell Res 30（12）：1136-1139，2020; Yip等人，Nature 587（7832）：157-161，2020）。在过去的5年里，它已被用作评估现代冷冻显微镜和加工方法的测试对象。与脱铁铁蛋白相反，细菌分泌系统是多蛋白质复合物的典型实例，其由于其与小分子、蛋白质和DNA转运到细胞外空间或靶细胞中有关的功能而表现出高柔性。这使得它们的结构表征极具挑战性（Barlow，Methods Mol Biol 532：397-411，2009; Costa等人，Nat Rev Microbiol 13：343-359，2015）。冷冻电子显微镜（EM）是揭示其空间组织和功能修饰的最可行的方法。在过去的十年中，结构冷冻电镜已被广泛用于分析生物复合物，包括多个组分，并不适合结晶（Lyumkis，J Biol Chem 294：5181-5197，2019;奥尔洛娃和Saibil，Methods Enzymol 482：321-341，2010;奥尔洛娃和Saibil，Chem Rev 111（12）：7710-7748，2011）。在这篇综述中，我们将描述用于冷冻EM的样品制备的基础，数字数据收集的原理，以及图像分析的后勤学，其重点是重建小型和大型生物复合体所需的共同步骤，以及将其结构细化到接近原子分辨率。通过IV型分泌系统的EM分析的例子来说明处理的流程。
Structural studies of bio-complexes using single particle cryo-Electron Microscopy (cryo-EM) is nowadays a well-established technique in structural biology and has become competitive with X-ray crystallography. Development of digital registration systems for electron microscopy images and algorithms for the fast and efficient processing of the recorded images and their following analysis has facilitated the determination of structures at near-atomic resolution. The latest advances in EM have enabled the determination of protein complex structures at 1.4–3 Å resolution for an extremely broad range of sizes (from ~100 kDa up to hundreds of MDa (Bartesaghi et al., Science 348(6239):1147–1151, 2015; Herzik et al., Nat Commun 10:1032, 2019; Wu et al., J Struct Biol X 4:100020, 2020; Zhang et al., Nat Commun 10:5511, 2019; Zhang et al., Cell Res 30(12):1136–1139, 2020; Yip et al., Nature 587(7832):157–161, 2020; https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year)). In 2022, nearly 1200 structures deposited to the EMDB database were at a resolution of better than 3 Å (https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year).To date, the highest resolutions have been achieved for apoferritin, which comprises a homo-oligomer of high point group symmetry (O432) and has rigid organization together with high stability (Zhang et al., Cell Res 30(12):1136–1139, 2020; Yip et al., Nature 587(7832):157–161, 2020). It has been used as a test object for the assessments of modern cryo-microscopes and processing methods during the last 5 years. In contrast to apoferritin bacterial secretion systems are typical examples of multi protein complexes exhibiting high flexibility owing to their functions relating to the transportation of small molecules, proteins, and DNA into the extracellular space or target cells. This makes their structural characterization extremely challenging (Barlow, Methods Mol Biol 532:397–411, 2009; Costa et al., Nat Rev Microbiol 13:343–359, 2015). The most feasible approach to reveal their spatial organization and functional modification is cryo-electron microscopy (EM). During the last decade, structural cryo-EM has become broadly used for the analysis of the bio-complexes that comprise multiple components and are not amenable to crystallization (Lyumkis, J Biol Chem 294:5181–5197, 2019; Orlova and Saibil, Methods Enzymol 482:321–341, 2010; Orlova and Saibil, Chem Rev 111(12):7710–7748, 2011).In this review, we will describe the basics of sample preparation for cryo-EM, the principles of digital data collection, and the logistics of image analysis focusing on the common steps required for reconstructions of both small and large biological complexes together with refinement of their structures to nearly atomic resolution. The workflow of processing will be illustrated by examples of EM analysis of Type IV Secretion System.