Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size (Ϸ85 nm envelope diameter). The viral envelope exhibits an unusual thickness (7.8 ؎ 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be extensively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of Ϸ4 nm. Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confirm all the general features and global architecture observed for MHV. We propose a general model for the structure of the coronavirion in which our own and published observations are combined.coronaviruses ͉ enveloped viruses ͉ plus-stranded RNA viruses ͉ transmissible gastroenteritis coronavirus

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.

Using transmission electron microscopy, automated data collection, and image stitching, biological specimens as large as one square millimeter can be ultrastructurally mapped at nanometer resolution.

No abstract

No abstract