Electron cryomicroscopy
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.
Docking crystal structures into cryo-EM mapsDocking 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[2]. Subsequently, many methods, including docking while allowing flexibility in the monomers, have been developed[3].
ResolutionResolution
In 2018, the median resolution of cryo-EM structures deposited in the Protein Data Bank was 3.8 Å (improved from 4.3 Å in 2016)[4]. For comparison, the median resolution of X-ray crystallographic entries in the PDB has been 2.0 Å for many years[4].
VideosVideos
- 3 min video illustrating the principles of cryo-EM.
- 2017 Nobel laureate Richard Henderson explains the history of cryo-EM in this 5 min video.
- Looking at Molecules: The electron cryo-microscopy revolution at The MRC LMB, 7 min.
- Cryo-EM17, a series of 5 lectures, about 45 min to 1 hour each, by experts at the Medical Research Council, Laboratory of Molecular Biology.
- A series of dozens of videos going into depth on the methodology of cryo-EM by Grant Jensen at CalTech starts with Welcome to Cryo-EM. Individual videos range from roughly 10 to 30 min each. The whole series adds up to about 14 hours.
- Movie of a vacuolar ATPase proton pump motor, based on cryo-EM structures obtained in multiple rotation states[5].
Protein IdentificationProtein 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 pili fibers made by bacteria, notably Geobacter sulfurreducens. Cryo-EM structure revealed that some of these pili are assembled from C-type cytochrome OmcS[6][7]. This was a surprise since these pili had long been thought to be assembled from a completely different protein, pilA.
RedirectsRedirects
Electron cryo-microscopy, Cryo-electron microscopy and Cryo-EM redirect to this page.
Notes and ReferencesNotes and References
- ↑ 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.
- ↑ 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
- ↑ 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
- ↑ 4.0 4.1 See Cryo-EM Resolution compared with X-ray diffraction resolution: tinyurl.com/method-vs-resolution.
- ↑ 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
- ↑ Wang, F. et al., in press in Cell.
- ↑ Preprint Structure of a cytochrome-based bacterial nanowire, Filman et al., December 2018.