Bringing Structure to Cell Biology with Cryo-Electron Tomography

Abstract

Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure-function relationships in their native environment and becoming a tool for discovering new biology.

Keywords: biophysical modeling; cryo-electron tomography; in situ structural biology; molecular architecture; quantitative cell biology.

Figure 1

Cryo-FIB-ET workflow. (a) Adherent cells can be cultured on EM grids, allowing unique lab-on-a-grid experiments. (b) A thin window or lamella is carved into an adherent grid grown on an EM grid via an FIB under cryogenic conditions. Expansion joints (trenches) on either side of the lamella are made to improve mechanical stability by offering points to allow flexing. (c) The lamella is imaged by TEM under cryogenic conditions at multiple tilt angles to facilitate a 3D reconstruction of the molecules within the lamella. (d) The complex and heterogeneous interior of a cell can be imaged at the highest achievable spatial resolution, resolving individual macromolecules (ribosomes, the NPC, nucleosomes), filaments (microtubules, actin, lamin), and organelles (mitochondria, ER, nucleus, Golgi). Abbreviations: cryo-FIB-ET, cryo-focused ion beam milling and electron tomography; EM, electron microscopy; ER, endoplasmic reticulum; FIB, focused ion beam; NPC, nuclear pore complex; TEM, transmission electron microscopy. Figure copyright 2023 Dorotea Fracchiolla, adapted with permission.

Figure 2

Data collection methods. What data collection method is best for your data? It depends. (a) In the dose-symmetric tilt series, data collection alternates between positive and negative tilts. (b) In continuous fast tilting, data are continuously acquired. (c) In hybrid STA-SPA, a high-dose image is collected at 0°, followed by implementation of the dose-symmetric method. (d) In beam-image shift, the beam is shifted from a central focus and tracking to targeted areas of acquisition, facilitating the collection of multiple tilt series simultaneously. (e) In a montage collection series, the entire lamella is imaged to create one large tomogram. (f) In 2D template matching, one single projection image is acquired at 0°, and high-resolution features of a target are matched to the data. Abbreviations: ET, electron tomography; SPA, single-particle analysis; STA, subtomogram analysis.

Figure 3

Cryo-ET by the numbers. The size and cellular abundance of a given macromolecule (or its local concentration) are relevant when estimating how many lamellae, tomograms, and particles you will need to perform STA. Not surprisingly, the more abundant protein complexes (e.g., ribosomes, nucleosomes) will have a greater ratio of particles to tomograms compared to less abundant complexes (e.g., the NPC). For the number of NPCs in yeast, see Reference ; particle numbers and estimated resolution from EMDB 11373 (166). For the number of NPCs in mammals, see Reference ; particle numbers and estimated resolution from EMDB 12814 (126). For the number of nucleosomes in the mammalian nucleus, see Reference ; particle numbers and estimated resolution from EMDB 6949 (23). For the number of bacterial ribosomes, see References and ; BioID 106861; particle numbers and estimated resolution from EMDB 11650 (137). For the number of mammalian ribosomes, see Reference ; BioID 106861; particle numbers and estimated resolution from Reference and EMDB XXXX (yet to be released). COPII particle numbers and estimated resolution from EMDB 3720 (38). Arp2/3 particle numbers and estimated resolution from EMDB 11869 (48). Abbreviations: ET, electron tomography; NPC, nuclear pore complex; S. pombe, Saccharomyces pombe; STA, subtomogram analysis; WT, wild type.