Cryo-Electron Tomography Studies of Cell Systems
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CTURE OF MACROMOLECULAR COMPOUNDS
Cryo-Electron Tomography Studies of Cell Systems R. A. Kamyshinskya,b, Y. M. Chesnokova,b, and A. S. Orekhova,b,* a
b
National Research Center “Kurchatov Institute,” Moscow, 123182 Russia Shubnikov Institute of Crystallography, Federal Research Center “Crystallography and Photonics,” Russian Academy of Sciences, Moscow, 119333 Russia *e-mail: [email protected] Received February 5, 2020; revised March 5, 2020; accepted March 17, 2020
Abstract—Cryo-electron tomography is a powerful tool for determining the three-dimensional structures of macromolecular complexes in their natural environment. The minimization of external impacts on the investigated objects, possibility of conducting in vitro and in cellulo experiments under close-to-native conditions, and high spatial resolution of the obtained three-dimensional reconstructions make cryo-electron tomography one of the most promising methods for studying a large class of objects in the fields of structural biology and visual proteomics. The main aspects of cryo-electron tomography are discussed as applied to studies of cell systems. DOI: 10.1134/S1063774520050090
INTRODUCTION The development of methods of cryo-electron microscopy (cryo-EM), especially pronounced in the last years [1], has significantly increased the number of macromolecular structures deciphered with a nearatomic resolution. The best known cryo-EM method, single particle analysis (SPA), has become popular as a powerful tool for studying single proteins and protein complexes in the near-native state. The name of this method reflects its main scenario, i.e., study of multiple copies of single protein macromolecules with a mass between 100 kDa–100 mDa [2] in different orientations, averaging the data (tens of thousands of images of the object projections), and reconstructing a high-resolution three-dimensional structure on their basis. Simultaneously, the method of cryo-electron tomography (cryo-ET) has been significantly developed. This technique provides a unique opportunity for direct visualization of molecular structures in their natural functional environment [3]. This method is mainly aimed at studying macromolecular complexes with variable morphology, as well as viruses, bacteriophages, and cells. Cryo-ET is applied to objects for which structural information cannot be obtained by other methods for some reasons, for example, in view of their non-crystallizability, large size, and difficulties with extracting and cleaning samples. The use of cryo-ET, in turn, makes it possible to study the complexes that are difficult to reproduce and extract directly in cells [4], as well as their conformations, mutual orientations, and interactions. As a rule, less than 1000 particles of an object of interest are used to process data and obtain
high-resolution structures using the cryo-ET technique. Thus, at present, cryo-ET fills the gap in physical dimensions of investigated objects and the spatial resolution of the data between super-resolution optical microscopy and
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