Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

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 cryotomography 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.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

“…Subvolumes can be aligned, classified, and averaged using the PEET package or EMAN2 (Step 64). Alternatively, the tilt series 2D images and the 3D volumes can be ported into any other available software package, such as Relion 17 , pyTom 18 , jsubtomo 19 , Bsoft 20 , or Dynamo 21 .…”

Cryo-electron tomography investigation of serum albumin-camouflaged tobacco mosaic virus nanoparticles

“…32,33 The resulting average SA density was then placed into the refined SA positions within the tomogram to visualize how SA is oriented on the TMV surface. Fig.…”

“…These densities were analyzed with the Jsubtomo software package 32 using a 20 Å filtered map of the SA crystal structure 33 (PDB: 1AO6) as a template map. Refined SA positions were selected for which Jsubtomo found dimer related orientations of the SA template in the cropped cryoEM density regions.…”

“…These density segments were analyzed with the Jsubtomo software package. 32 An SA-TMV template map was generated with an 8.8 nm diameter sphere, representing the same volume as SA, positioned 2.4 nm away from the surface of a 20 Å filtered map of six helical turns of TMV (PDB: 4UDV; Fig. 5A).…”

Cryo-EM of viruses and vaccine design

“…The intermolecular interactions between the asymmetrical units of the icosahedral capsid provide a level of order that increases the accuracy of alignment, a feature also relevant for viruses with helical capsids (10,11). In contrast, the outer membranes of enveloped viruses, such as influenza, Ebola, and HIV, display their envelope proteins in a highly disordered arrangement, and structure determination of envelope glycoproteins requires the use of subvolume averaging methods, which have only produced low-resolution structures so far (12,13).…”