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. 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

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. Subsequently, many methods, including docking while allowing flexibility in the monomers, have been developed.

ResolutionResolution

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). For comparison, the median resolution of X-ray crystallographic entries in the PDB has been 2.0 Å for many years. 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.

Temperatures (B factors)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".

VideosVideos

• What is Cryo-Electron Microscopy (Cryo-EM)? UC San Francisco (UCSF) video explaining what is Cryo-EM, 2.5 min.
• 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 10 lectures, ~45-60 min each, by experts at the Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK
• A series of 48 in depth videos on the methodology of Cryo-EM by Grant Jensen at CalTech starting with Welcome to Cryo-EM. Individual videos range from roughly 10-30 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 by Zhao, Benlekbir & Rubinstein (2015), 1.5 min.
• Video illustrating the principles of Cryo-EM by Gabriel Lander (Scripps Research), 3 min.

Protein IdentificationProtein Identification

In the 21^st 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. This was a surprise since they had long been thought to be assembled from a completely different protein, pilA.

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