Abstract
The architecture of protein assemblies and their remodeling during physiological processes is fundamental to cells. Therefore, providing high-resolution snapshots of macromolecular complexes in their native environment is of major importance for understanding the molecular biology of the cell. Cellular structural biology by means of cryo-electron tomography (cryo-ET) offers unique insights into cellular processes at an unprecedented resolution. Recent technological advances have enabled the detection of single impinging electrons and improved the contrast of electron microscopic imaging, thereby significantly increasing the sensitivity and resolution. Moreover, various sample preparation approaches have paved the way to observe every part of a eukaryotic cell, and even multicellular specimens, under the electron beam. Imaging of macromolecular machineries at high resolution directly within their native environment is thereby becoming reality. In this review, we discuss several sample preparation and labeling techniques that allow the visualization and identification of macromolecular assemblies in situ, and demonstrate how these methods have been used to study eukaryotic cellular landscapes.
Highlights
Understanding the basic mechanisms of life at the molecular level is one of the fundamental aims of cell and structural biology
Albert et al impressively showed that two distinct structural classes of 26S proteasomes crowd around nuclear pore complexes (NPCs), where they are presumably involved in protein quality control and degradation of misfolded proteins [26]
The detergent disrupts the plasma membrane and removes most of the soluble components of the cytoplasm, while only insoluble elements such as intermediate filaments (IFs) and the nucleus remain, which can be analyzed by cryo-electron tomography (cryo-ET) [30]
Summary
Understanding the basic mechanisms of life at the molecular level is one of the fundamental aims of cell and structural biology. Traditional structural biology techniques are restricted to the analysis of macromolecules in isolation, and determine their molecular structure one by one. These studies have yielded an impressive amount of mechanistic information that has revolutionized our understanding of basic processes in cells [1,2]. Since its first application to eukaryotic cells [5], cryo-electron tomography (cryo-ET) has become a pivotal approach in cell biology [6,7,8,9,10,11], microbiology [12,13,14,15,16,17], and virology [18,19,20,21,22], as it is the only available technique that allows structure determination of macromolecular complexes at close-to-native conditions directly within a cell [3,23,24]. We will explore ways to localize and identify specific elements of interest in the crowded cellular environment
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