Abstract
Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein ‘spikes’ is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.
Highlights
Electron cryo-tomography is an electron cryo-microscopy imaging technique allowing the calculation of a three-dimensional (3D) reconstruction of complex biological specimens
We demonstrate the application of the sub-tomogram averaging workflow outlined above for the envelope glycoprotein complex of Bunyamwera virus (Orthobunyavirus, Bunyaviridae) using a previously published data set[24]
Knowledge of viral glycoprotein spike structure on the virion membrane is essential for understanding virus replication and developing therapeutics to treat and prevent infection
Summary
Electron cryo-tomography is an electron cryo-microscopy imaging technique allowing the calculation of a three-dimensional (3D) reconstruction of complex biological specimens. As a result of limited sample tilt geometry during data collection, some views of the object remain absent, leading to a so-called ‘missing wedge’ artifact in the tomographic volume Both of these limitations can be overcome if the tomographic volume contains repeating identical structures, such as macromolecular complexes, that can be successfully averaged[9,10,11,12]. While macromolecular crystallography is the technique of choice for high-resolution (usually better than 4 Å) structural analysis of individual viral glycoproteins and their complexes, the X-ray structures resulting from this method are of proteins isolated from the natural membranous environment on the virion. Input data for this protocol is a set of tomographic reconstructions of the virions. The names of individual programs are given in italics and file formats are denoted with uppercase filename extensions
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