Cryo-electron tomography reveals the structural biology of native macromolecules.

Single-particle cryo-electron microscopy (cryo-EM) has emerged as a transformative approach for determining high-resolution structures of proteins and nucleic acids. Methods for solving structures by cryo-EM have developed and matured, increasing the uptake of this approach by the broader research community.

Ribosomes in a cryo-electron tomogram. Adapted with permission from Schaffer et al. Nat. Methods 16, 757–762 (2019), Springer Nature.

However, the advantages of electron microscopy for structural biology go well beyond single-particle cryo-EM. For resolving structures as they occur in their native cellular context, cryo-electron tomography (cryo-ET) comes to the fore.

In cryo-ET, a tilt series of 2D images of a sample are taken using a transmission electron microscope; each image is taken at a slightly different angle, after which computational methods are used to create a tomographic, 3D reconstruction of the sample. This 3D reconstruction represents a snapshot of the cell and can capture transient and rare events that can be missed when using purified components. By combining averaging tools from single-particle cryo-EM with images obtained with cryo-ET, structures can currently be obtained with 10–30 Å resolution. These tomograms can also be fitted with high-resolution structures obtained using other approaches to create detailed models of structures and complexes as they occur in cells.

Although cryo-ET has already yielded rich structural information in cellular contexts, better methods are still needed to realize its full potential. Current implementations are often limited to thin samples, such as bacteria; its application has been more limited in mammalian cells. But improved methods for focused ion beam milling of samples for cryo-ET, for example, can give access to thicker cells and tissues (Nat. Methods 12, 634–636, 2015; Nat. Methods 16, 757–762, 2019; Nat. Methods https://doi.org/10.1038/s41592-019-0630-5, 2019).

There are also challenges of image analysis. The ‘missing wedge’ plagues many tomographic approaches and reduces resolution; it arises because images cannot be acquired for all tilt angles. Computational approaches are making exciting headway to address this problem (J. Struct. Biol. 206, 183–192, 2019). Another challenge is annotating particles of interest from the crowded intracellular environment, with computational approaches greatly facilitating the process (Nat. Methods 14, 983–985, 2017). Additional processing and computational workflows further improve cryo-ET (Nat. Methods 15, 955–961, 2018; Nat. Methods 16, 1161–1168, 2019).

We anticipate future methods development will improve throughput, sample preparation, analysis, and achievable resolution of cryo-ET.