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This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.

Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.electron cryomicroscopy | matrix protein | ribonucleoprotein particles hemagglutinin | neuramindase

The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions. In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O-acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O-acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, alpha-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.

The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.

SignificanceX-ray crystallography studies of soluble ectodomains of influenza hemagglutinins (HA) have previously revealed details of their two functions in virus infection: receptor binding and membrane fusion. We have now used cryo-EM to determine the structures of full-length hemagglutinin solubilized in detergent, alone, and in complex with the Fab of an infectivity neutralizing antibody. We observe the structure of the region that anchors HA in the virus membrane as a bundle of three α-helices that are joined to the ectodomain by flexible linkages. The Fab binds near the linkages and restricts their flexibility. This may indicate that the flexibility is required for HA-mediated membrane fusion and that interference with it represents a mechanism for neutralizing virus infectivity.