Electron cryomicroscopy

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Single-particle electron cryomicroscopy (cryo-EM) has become an important method for determining macromolecular structures. It is the basis for the 2017 Nobel Prize in Chemistry. Although resolution is usually poorer than that obtained by X-ray crystallography, cryo-EM has the great advantage of not requiring crystallization[1]. Cryo-EM is particularly suited to determination of the structures of large complexes containing multiple proteins or nucleic acids, often the most difficult to crystallize.

For a quick overview of the method, see this silent 3 min video by Gabe Lander. For a historical view of the development of modern cryo-EM, see this scientific background for the 2017 Nobel prize. For a more detailed discussion, see this primer[2]

Contents

Docking crystal structures into cryo-EM maps

Early studies showed that docking of monomer crystal structures into even poor-resolution (e.g. 15 Å) cryo-EM maps of larger assemblies could reliably predict structure[3]. Subsequently, many methods, including docking while allowing flexibility in the monomers, have been developed[4].

Resolution

Temperatures (B factors)

PDB files for models determined by cryo-EM often specify values in the temperature/B factor field. However, a 2017 analysis concluded that "the treatment of the atomic displacement (B) factors was meaningless in almost all analyzed cryo-EM models"[6].

Videos

Protein Identification

In the 21st century, the amino acid sequence identity of a protein is nearly always known before its structure is determined. This is because the gene is usually cloned, the protein expressed and purified, prior to crystallization. However, it is also possible to purify macromolecular assemblies in which the proteins are not identified in advance, and determine their structures by cryo-EM. If sufficient resolution is achieved (~3.5 Å or better), candidate amino acid sequences can be matched, or excluded, as components of the structure. Thus, typically combined with mass spectrometry, cryo-EM can help identify which proteins are present in a structure[8].

An example is the electrically-conductive protein nanowires made by bacteria, notably Geobacter sulfurreducens. Cryo-EM structure revealed that some of these fibers are assembled from C-type cytochrome OmcS[9][10]. This was a surprise since they had long been thought to be assembled from a completely different protein, pilA.

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Notes and References

  1. ↑ Obtaining highly-ordered crystals is perhaps the major obstacle to determination of structure by X-ray diffraction. Less than half of cloned, expressed, purified proteins are sufficiently soluble for structure determination. Of these, diffraction-quality crystals are obtained for only about one in five. See Structural Genomics Progress, 2011.
  2. ↑ Cheng Y, Grigorieff N, Penczek PA, Walz T. A primer to single-particle cryo-electron microscopy. Cell. 2015 Apr 23;161(3):438-449. doi: 10.1016/j.cell.2015.03.050. PMID:25910204 doi:http://dx.doi.org/10.1016/j.cell.2015.03.050
  3. ↑ Roseman AM. Docking structures of domains into maps from cryo-electron microscopy using local correlation. Acta Crystallogr D Biol Crystallogr. 2000 Oct;56(Pt 10):1332-40. PMID:10998630
  4. ↑ Kim DN, Sanbonmatsu KY. Tools for the cryo-EM gold rush: going from the cryo-EM map to the atomistic model. Biosci Rep. 2017 Dec 5;37(6). pii: BSR20170072. doi: 10.1042/BSR20170072. Print, 2017 Dec 22. PMID:28963369 doi:http://dx.doi.org/10.1042/BSR20170072
  5. ↑ 5.0 5.1 See cryo-EM Resolution compared with X-ray diffraction resolution: tinyurl.com/method-vs-resolution.
  6. ↑ Wlodawer A, Li M, Dauter Z. High-Resolution Cryo-EM Maps and Models: A Crystallographer's Perspective. Structure. 2017 Oct 3;25(10):1589-1597.e1. doi: 10.1016/j.str.2017.07.012. Epub, 2017 Aug 31. PMID:28867613 doi:http://dx.doi.org/10.1016/j.str.2017.07.012
  7. ↑ Zhao J, Benlekbir S, Rubinstein JL. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature. 2015 May 14;521(7551):241-5. doi: 10.1038/nature14365. PMID:25971514 doi:http://dx.doi.org/10.1038/nature14365
  8. ↑ Ho CM, Li X, Lai M, Terwilliger TC, Beck JR, Wohlschlegel J, Goldberg DE, Fitzpatrick AWP, Zhou ZH. Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu. Nat Methods. 2019 Nov 25. pii: 10.1038/s41592-019-0637-y. doi:, 10.1038/s41592-019-0637-y. PMID:31768063 doi:http://dx.doi.org/10.1038/s41592-019-0637-y
  9. ↑ Wang F, Gu Y, O'Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Cell. 2019 Apr 4;177(2):361-369.e10. doi: 10.1016/j.cell.2019.03.029. PMID:30951668 doi:http://dx.doi.org/10.1016/j.cell.2019.03.029
  10. ↑ Filman DJ, Marino SF, Ward JE, Yang L, Mester Z, Bullitt E, Lovley DR, Strauss M. Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire. Commun Biol. 2019 Jun 19;2:219. doi: 10.1038/s42003-019-0448-9. eCollection 2019. PMID:31240257 doi:http://dx.doi.org/10.1038/s42003-019-0448-9

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