Abstract
Cryo-electron tomography (CET) is uniquely suited to obtain structural information from a wide range of biological scales, integrating and bridging knowledge from molecules to cells. In particular, CET can be used to visualise molecular structures in their native environment. Depending on the experiment, a varying degree of resolutions can be achieved, with the first near-atomic molecular structures becoming recently available. The power of CET has increased significantly in the last 5 years, in parallel with improvements in cryo-EM hardware and software that have also benefited single-particle reconstruction techniques. In this review, we cover the typical CET pipeline, starting from sample preparation, to data collection and processing, and highlight in particular the recent developments that support structural biology in situ. We provide some examples that highlight the importance of structure determination of molecules embedded within their native environment, and propose future directions to improve CET performance and accessibility.
Highlights
The electron microscope provides a powerful tool to understand biological processes over a wide range of scales, from the determination of molecular structures to the characterisation of cell morphology (Figure 1).For decades, electron microscopy (EM) of stained and sectioned cells helped define cell morphology and ultrastructure, understand the function of organelles, and identify the aetiology of many diseases
Thanks to the adaptation of dose-compensation schemes developed for detector devices (DEDs) in single-particle cryo-EM [35], total electron doses exceeding 100 electrons/A2 have been used to obtain cryo-tomograms with higher signal-to-noise ratios (SNR), in the knowledge that inflicted damage at high resolutions can be later removed as a post-processing step
We have provided a review of the steps involved in Cryo-electron tomography (CET), from sample preparation to data processing and interpretation
Summary
The electron microscope provides a powerful tool to understand biological processes over a wide range of scales, from the determination of molecular structures to the characterisation of cell morphology (Figure 1).For decades, electron microscopy (EM) of stained and sectioned cells helped define cell morphology and ultrastructure, understand the function of organelles, and identify the aetiology of many diseases. Great advances have followed upon the development of techniques to prepare cells for visualisation in cryo-conditions, including CEMOVIS (cryo-EM of vitrified specimens) [2] and more recently focused ion beam (FIB)– scanning electron microscopy (SEM) [3,4,5], which allow preservation of the native cellular structures. Cryo-electron tomography (CET) and subtomogram averaging (STA) are fast-developing techniques that allow structure determination in situ, bridging the gap across biological scales.
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