Electron cryomicroscopy

From Proteopedia

Jump to: navigation, search

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 more detailed discussion, see this primer[2]


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].


In 2018, the median resolution of cryo-EM structures deposited in the Protein Data Bank was 3.8 Å (improved from 4.2 Å in 2016)[5]. For comparison, the median resolution of X-ray crystallographic entries in the PDB has been 2.0 Å for many years[5]. When resolution improves by a factor of 2, the available data (to support the coordinate model) goes up by a factor of 8. For example, a 2.4 Å resolution structure is a great improvement over a 3.0 Å resolution structure because the number of available measurements doubles.

A direct comparison between the quality of cryo-EM structures and crystal structures is not possible. In crystal structures, the electron density is calculated from measured structure factors and from calculated phases (in the most extreme case, half of the information is not available from experiment). Even in cases were there are experimental phases (e.g. from multiple isomorphous replacement), these are typically not available to the full resolution. Especially at resolutions below 4 Å, X-ray structures are prone to model bias in the absence of experimental phases. Cryo-EM does not have this limitation, so low resolution structures can still carry reliable information, for instance about conformational changes of known structures. For example, a 4 Å crystal structure solved by molecular replacement (i.e. no experimental phases) is not as reliable as a 4 Å cryo-EM structure.


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.

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[7][8]. This was a surprise since they had long been thought to be assembled from a completely different protein, pilA.


Electron cryo-microscopy, Cryo-electron microscopy and Cryo-EM redirect to this page.

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. 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
  7. 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
  8. 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

Proteopedia Page Contributors and Editors (what is this?)

Eric Martz, Karsten Theis

Personal tools