Cryo-EM: expanding the reach of structural biology

Catherine L. Lawson
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Electron cryo-microscopy (cryo-EM) is rapidly becoming the method of choice to elucidate many important 3D structures in molecular and cellular biology, expanding the reach of structural biology well beyond materials that can be crystallized. I provide below a very brief summary of how we arrived at this point. The timeline described here follows in part a more extensive review that will shortly be published as part of a special collection celebrating the 50th anniversary of the Protein Data Bank (PDB) (Chiu et al., 2021).

EM scientists began investigating the overall shapes of biological materials in the mid-20th century. Early successes involved specimens with high internal symmetries – 2D periodicities, icosahedral viruses and helical assemblies. Symmetry enables averaging over many individual imaged subunits to obtain a final 3D reconstruction (density map). Experimental measurements typically include both electron diffraction (spots or layer lines) and direct images (for phasing). The 1982 Nobel Prize in Chemistry recognized the development of electron crystallography (Klug, 2010).

Minimizing radiation damage of biological materials on exposure to high-energy electrons was a major technological challenge. Early strategies involved low-dose exposure procedures and chemical embedding. Methods to vitrify and maintain specimens at cryogenic temperatures began to be developed in the mid-1970s, leading to substantial improvements in the collected data quality (Baker & Rubinstein, 2010).

The first 3DEM Gordon Research Conference was held in 1985 (see photo archived at https://www.ebi.ac.uk/pdbe/emdb/genealogy.html). The inaugural meeting, chaired by Wah Chiu, firmly established cryo-EM as a serious endeavor with a dedicated community of research scientists. The 3DEM GRC continues to play a critical role in sharing recent developments in the field. Meetings are held annually (outside of pandemics) with venues alternating between the US, Europe and Asia.

The 1990s and 2000s yielded many technological innovations, gradually increasing cryo-EM’s overall impact (Henderson, 2004). Microscopes with field electron gun sources became common in academic laboratories, yielding improved image signal-to-noise and spatial resolution. Keen interest in asymmetric (e.g. ribosomes) and lower-symmetry complexes (e.g. chaperonins) sparked the intensive development of single-particle reconstruction methods. Tomographic and sub-tomogram averaging methods were developed, enabling investigations of in situ materials in cellular and tissue samples such as muscle fibers. Software developers created packages that automated data collection, image classification and reconstruction. Some embraced electronic CCD detectors for their ability to automate data collection, but film still continued to be prized for its high sensitivity.

Crystallography and cryo-EM studies started to be combined in complementary ways. A common approach was to fit high-resolution crystal structures into low-resolution maps of large macromolecular complexes. Alternatively, low-resolution cryo-EM maps could provide critical phasing information for crystal-structure determination.

In this period, cryo-EM results were appearing with increasing regularity in the scientific literature. The community recognized a need to archive 3D reconstructions (density maps) and make them publicly accessible. The Electron Microscopy Data Bank (EMDB) was established in 2002 by Kim Henrick at the European Bioinformatics Institute (EBI) for this purpose. Soon after, and with input from many cryo-EM experts, Henrick and Helen Berman (Research Collaboratory for Structural Bioinformatics; RCSB PDB) spearheaded the development of a dictionary of data terms for cryo-EM experiments that could be utilized by both the EMDB and the PDB. In 2006, an NIH-funded international project was founded by Chiu, Henrick and Berman to provide a one-stop-shop for deposition and access to cryo-EM data and to serve as a community resource for news, events, software tools, data standards, challenges and validation methods [EMDataResource (Lawson et al., 2020)].

Seven years ago, cryo-EM had its famous “resolution revolution” (Kuhlbrandt, 2014). A key innovation was the introduction of new direct electron detectors with improved sensitivity and high speed, permitting the collection of movies. Post-imaging alignment of movie frames enabled researchers to overcome the previous limitations arising from specimen drift, yielding maps with much higher levels of structural detail (Fig. 1). Cryo-EM scientists have also learned how to computationally separate different classes of particles measured within the same set of images, yielding multiple reconstructions that can differ in composition and/or conformation. These innovations have substantially extended the reach of cryo-EM across a wide variety of specimens that are not readily crystallized (Fig. 2). The rapidly rising profile of cryo-EM in structural biology was internationally recognized by the 2017 Nobel Prize in Chemistry.

[Figure1]Figure 1. Shifting resolution profiles for cryo-EM maps archived in EMDB, plotted by map release year. The 2021 data are through 3 February. Source: https://www.emdataresource.org/statistics.html

 

[Fig. 2]Figure 2. Sampling of cryo-EM maps recently released in EMDB. Maps include several membrane-bound and soluble enzymes, a light-harvesting complex, a coronavirus spike glycoprotein and an icosahedral virus. Source: https://www.emdataresource.org/emdlist=released:2021

The recent pandemic has demonstrated the growing utility of cryo-EM. Important structural data about the circulating virus was produced very early on, with the first SARS-CoV-2 spike glycoprotein structure released on 26 February 2020 (Wrapp et al., 2020). A year later, more than 270 spike-containing map entries have been archived in the EMDB, representing the trimer, either alone or in complexes with antibody or receptor fragments. More than 60 maps are also available for other SARS-CoV-2 virus components such as polymerase and ribonucleoprotein.

In this rapid growth phase of cryo-EM, it is necessary to establish rigorous methods to validate the maps and associated models. EMDataResource promotes public awareness of this necessary step in structure determination, as is done in crystallography by sponsoring regular community Challenge activities and organizing and participating in community Task Forces (Lawson et al., 2020, 2021; Chiu et al., 2021).

References

Baker, L. A. & Rubinstein, J. L. (2010). Methods Enzymol. 481, 371–388.

Chiu, W., Schmid, M. F., Pintilie, G. & Lawson, C. L. (2021). J. Biol. Chem. In the press.

Henderson, R. (2004). Q. Rev. Biophys. 37, 3–13.

Klug, A. (2010). Annu. Rev. Biochem. 79, 1–35.

Kühlbrandt, W. (2014). Science, 343, 1443–1444.

Lawson, C. L., Berman, H. M. & Chiu, W. (2020). 'Evolving data standards for cryo-EM structures', Struct. Dyn. 7: 014701.

Lawson, C. L. A., Kryshtafovych, A., Adams, P. V., Afonine, M. L., Baker, B. A., Barad, P., Bond, T., Burnley, R., Cao, J., Cheng, G., Chojnowski, K., Cowtan, K. A., Dill, F., DiMaio, F., Farrell, J. S., Fraser, M. A., Herzik, S. W. Jr, Hoh, J., Hou, J., Hung, M., Igaev, M., Joseph, D., Kihara, D., Kumar, S., Mittal, B., Monastyrskyy, M., Olek, M., Palmer, A., Patwardhan, A., Perez, J., Pfab, J., Pintilie, J. S., Richardson, P. B., Rosenthal, D., Sarkar, D., Schäfer, M. F., Schmid, G. F., Schröder, M., Shekhar, D., Si, A., Singharoy, G., Terashi, G., Terwilliger, A., Vaiana, L., Wang, Z., Wang, Z., Wankowicz, C. J., Williams, M., Winn, T., Wu, X., Yu, K., Zhang, K., Berman, H. M. & Chiu, W. (2021). Nat. Methods, 18, 156–164.

Wrapp, D. N., Wang, N., Corbett, J. A., Goldsmith, C. L., Hsieh, O., Abiona, O., Graham, B. S. & McLellan, J. S. (2020). Science, 367, 1260–1263.

 

Catherine L. Lawson is at the EMDataResource Project, Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, USA; cathy.lawson@rutgers.edu.


IUCr Journals have long championed the inclusion of research using techniques that complement crystallography, and recognised the spectacular growth in the adoption of cryo-EM by launching a new section of IUCrJ in 2016. Acta Cryst. D regularly publishes the proceedings of the Collaborative Computational Project for electron cryo-microscopy (CCP-EM) Spring Symposium, and together with Acta Cryst. F welcomes articles on cryo-EM as well as studies using other structural biology techniques.

15 March 2021

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