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Cryo-electron tomography of bacteria: progress, challenges and future prospects

Key Points

  • Cryo-electron tomography is an emerging method for three-dimensional (3D) imaging that bridges the gap between analyzing protein structure by X-ray crystallography and intact bacterial structure by light microscopy.

  • Electron tomography of bacterial cells has enabled the in situ visualization of subcellular components such as ribosomes, flagellar rotor complexes, cytoskeletal filaments and other large macromolecular complexes. Detection of small protein complexes, complexes that lack high intrinsic contrast, or those that do not form ordered arrays within the cell, is still rare despite the fact that most cellular proteins fall within these categories. Development of effective labelling technologies is essential to overcome this challenging imaging dilemma.

  • Electron tomography has yielded information of the highest quality on subcellular structure of bacteria that are < 500 nm thick or when thinner regions of thick cells, such as their poles, are analysed. A useful alternative strategy is to cut thick cells into thin sections, which can be imaged individually.

  • Although cryo-electron tomography is useful for imaging select protein complexes in a near-native state, tomography of plastic-embedded sections is also very useful in a number of instances. Although there is loss of structural detail, carefully prepared plastic-embedded sections can retain considerable integrity, and can offer quick detailed maps of the 3D membrane architecture.

  • The application of 3D averaging to electron tomographic data is a powerful strategy for determining the structure of molecular complexes in situ. Complexes with high intrinsic contrast, such as ribosomes, bacterial flagellar motors and ordered arrays such as the chemotaxis signalling complex have been successfully analysed. Discrete membrane receptor conformations can be discerned, providing proof of principle that the structural basis of signalling can be probed in whole cells.

Abstract

Recent advances in three-dimensional electron microscopy provide remarkable tools to image the interior of bacterial cells. Glimpses of cells at resolutions that are 1–2 orders of magnitude higher than those currently attained with light microscopy can now be obtained with cryo-electron tomography, especially when used in combination with new tools for image averaging. This Review highlights recent advances in this area and provides an assessment of the general applicability, current limitations and type of structural information that can be obtained about the organization of intact cells using tomography. Possible future directions for whole cell imaging are also discussed.

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Figure 1: From cell to tomogram.
Figure 2: Selected examples of findings from cryo-electron tomography of bacterial cells.
Figure 3: Two alternative strategies for three-dimensional reconstruction of thick cells.
Figure 4: Selected findings from cryo-electron microscopy of vitreous sections of high-pressure frozen bacterial cells.
Figure 5: Molecular structures of bacterial components in situ determined by three-dimensional averaging of individual volumes extracted from whole cell tomograms.

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Acknowledgements

This work was supported by funds from the intramural research program of the National Cancer Institute, National Institues of Health (NIH), USA.

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Supplementary information

Supplementary information S1 (figure)

Review of key findings from cryo-electron tomography of bacterial cells (see Table 1 in main text for complementary information) (PDF 960 kb)

Supplementary information S2 (figure)

Review of key findings from cryo-electron microscopy of vitreous sections of high-pressure frozen bacterial cells (see Table 2 in main text for complementary information). (PDF 840 kb)

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Glossary

Electron tomography

Strategy to obtain three-dimensional images of cells and other macromolecular assemblies using transmission electron microscopy.

Computerized axial tomography

A medical imaging method to obtain a three-dimensional image of the interior of an object by collecting a series of two-dimensional projection images which are digitally combined.

Vitrification and vitreous ice

Vitrification refers to the rapid freezing of biological samples at rates of greater than 105 kelvin per second that leads to the formation of vitreous or amorphous ice (in which the water molecules are randomly arranged like the atoms of glass) and hence prevents the formation of crystalline ice (in which the water molecules are arranged in an orderly pattern) which would otherwise disrupt biological structures.

Cryogen

A liquid such as liquid nitrogen, liquid ethane or liquid helium, that has a boiling point at low temperatures, typically below 100 K.

S-layer

A surface layer 5–25 nm thick that is part of the cell envelope of certain bacteria or archaea.

GroEL

A molecular chaperone found in a large number of bacteria that works in a complex with the related molecule GroES to mediate the proper folding of many cellular proteins.

Soft X-rays

Ionizing electromagnetic radiation that has a wavelength in the range 20 to 200 Å and energies of 12 to 120 keV that can be used to image thick biological specimens ranging from cells to thick tissues and organisms.

Synchrotron

A large machine in which charged sub-atomic particles are accelerated around a fixed circular path. Among many uses of the radiation generated by the synchrotron is its use for structure determination using X-ray crystallography.

Inelastic scattering

A mode of interaction of electrons with matter in a way that changes the kinetic energy of the incident electron.

Microtome

An instrument containing a sharp knife with a glass or diamond edge that is used to cut biological specimens into thin sections for their consequent analysis by light or electron microscopy.

Ion-abrasion scanning electron microscopy

An imaging technique that involves the iterative use of an ion beam to abrade away a surface layer of specified thickness, and the use of a scanning electron beam to image the newly exposed surface. The resulting stack of images is computationally processed to create a three-dimensional volume of the sample.

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Milne, J., Subramaniam, S. Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7, 666–675 (2009). https://doi.org/10.1038/nrmicro2183

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