Electron cryomicroscopy: Difference between revisions
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==Protein Identification== | ==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 | 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<ref>Preprint [https://www.biorxiv.org/content/early/2018/12/10/492645 Structure of a cytochrome-based bacterial nanowire], Filman ''et al.'', December 2018.</ref>. This was a surprise since these pili had long been thought to be assembled from a completely different protein, pilA. | 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<ref>Preprint [https://www.biorxiv.org/content/early/2018/12/10/492645 Structure of a cytochrome-based bacterial nanowire], Filman ''et al.'', December 2018.</ref>. This was a surprise since these pili had long been thought to be assembled from a completely different protein, pilA. | ||