Cryo-electron Microscopy Illuminates the Molecular Bases of Protein Synthesis Machinery

Abstract

Cryo-electron microscopy (cryo-EM) is a technique that enables the visualization of biological specimens under near-physiological conditions by rapidly freezing the samples and directly observing them at cryogenic temperatures using a transmission electron microscope. Biomolecular complexes in cells exert their functions while dynamically changing their structures. We focus on the ribosome, the protein synthesis machinery at the heart of the central dogma, and aim to elucidate the behavior of molecules across multiple scales by using cryo-electron microscopy. The development of cryo-EM has been driven by the accumulation of technological advances in two major methodologies. In single-particle analysis, purified biological specimens are embedded in vitreous ice, and three-dimensional structures are reconstructed from a large number of two-dimensional projection images by image processing. Cryo-electron tomography (cryo-ET) enables three-dimensional structural analysis of larger cellular regions, by imaging frozen specimens over a range of tilt angles, and is therefore well suited for studying structures beyond individual molecules targeted in single-particle analysis. However, electron beam penetration is limited by sample thickness. In the 2000s, the introduction of cryogenic focused ion beam–scanning electron microscopy (cryo-FIB-SEM) enabled precise micromachining of targeted regions of frozen specimens while preserving the vitrified state. Subsequent technological developments have greatly expanded the applicability of cryo-electron tomography (cryo-ET). Currently, in addition to structural analysis of the ribosome by single-particle analysis, we are also trying in situ visualization of ribosomes from diverse organisms using cryo-FIB-SEM. In this article, we provide an overview of the workflow from sample preparation to data acquisition in cryo-EM.