Imaging and visualizing SARS-CoV-2 in a new era for structural biology

Abstract

The SARS-CoV-2 pandemic has had a global impact and has put scientific endeavour in the spotlight, perhaps more than any previous viral outbreak. Fortuitously, the pandemic came at a time when decades of research in multiple scientific fields could be rapidly brought to bear, and a new generation of vaccine platforms was on the cusp of clinical maturity. SARS-CoV-2 also emerged at the inflection point of a technological revolution in macromolecular imaging by cryo-electron microscopy, fuelled by a confluence of major technological advances in sample preparation, optics, detectors and image processing software, that complemented pre-existing techniques. Together, these advances enabled us to visualize SARS-CoV-2 and its components more rapidly, in greater detail, and in a wider variety of biologically relevant contexts than would have been possible even a few years earlier. The resulting ultrastructural information on SARS-CoV-2 and how it interacts with the host cell has played a critical role in the much-needed accelerated development of COVID-19 vaccines and therapeutics. Here, we review key imaging modalities used to visualize SARS-CoV-2 and present select example data, which have provided us with an exceptionally detailed picture of this virus.

Keywords: SARS-CoV-2; X-ray crystallography; cryo-electron microscopy; cryo-electron tomography; multiscale imaging; structural biology.

Figure 1.

Recent technological advances have allowed us to image SARS-CoV-2 and its components on a continuous resolution spectrum up to the near-atomic scale. From left to right: a schematic of spike protein on the surface of cells as it would appear if visualized by fluorescent light microscopy; a schematic of a SARS-CoV-2 virion as it would appear if visualized by cryo-electron tomography; a cryo-EM reconstruction of the spike protein trimer (EMD-21374); a model based on the crystal structure of ACE2 (angiotensin-converting enzyme 2) in complex with the receptor binding domain (RBD) of spike protein (PDB 6M0J).

Figure 2.

SARS-CoV-2 and the cell. (a) Electron microscopy plays an important confirmatory role in diagnosis and determining subcellular localization. A false-colour transmission electron micrograph of SARS-CoV-2 isolated from a patient. Coronaviruses are approximately 80–120 nm in diameter [7]. (b) A false-colour scanning electron micrograph of an apoptotic cell (green) infected with SARS-CoV-2 isolated from a patient. The virions can be observed as small yellow spheres. (a) and (b) are adapted from images of ‘Novel Coronavirus SARS-CoV-2’ published by NIAID and licensed under CC BY 2.0 [8,9]. To view a copy of this license, visit https://creativecommons.org/licenses/by/2.0/. (c) Fluorescence microscopy can show subcellular localization. Co-localization of FLAG-tagged SARS-CoV-2 protein Orf6 with nuclear pore complex protein Nup358 in HEK293T cells as imaged by stimulated emission depletion (STED) super-resolution microscopy. Scale bar in the left-most panel is 5 µm. Scale bar in the magnified panels is 1 µm. This panel is adapted from ref. [10] and licensed under CC BY 4.0. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Figure 3.

A technological revolution in cryo-EM has opened new imaging frontiers. (a) Cryo-EM map of stabilized trimeric spike ectodomain (EMD-21375) fitted with its corresponding atomic model (PDB 6VSB). Inset shows a closer view of the correspondence between the map and the model. N-terminal domain, blue: RBD, purple, C-terminal domain, green; fusion peptide, red; HR1, yellow. (b) Cryo-EM map of the apo RNA-dependent RNA polymerase (EMD-11007) fitted with its corresponding atomic model (PDB 6YYT). Remdesivir is shown in purple, positioned at the end of the RNA product. Its position is based on the alignment of a remdesivir-bound structure (PDB 7BV2) with the apo model. A clipped perspective of the remdesivir binding site is highlighted in the inset. nsp7, blue; nsp8, green; polymerase domains: ‘fingers’, yellow; ‘palm’, orange; ‘thumb’, red.

Figure 4.

Cryo-electron tomography (cryo-ET) allows visualization in situ. (a) Three-dimensional volume rendering of a single virion derived from cryo-ET. The spike glycoprotein (purple) and viral ribonucleoprotein complexes (cyan) are subtomogram averages placed at their corresponding locations in the tomogram with vRNP orientations randomized. This panel is adapted from ref. [53] and licensed under CC BY 4.0. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. (b) Subtomogram averages of spike protein from the surface of virions. From left to right: closed conformation (EMD-11494), one RBD up (EMD-11495), two RBDs up (EMD-11496). RBDs in the up conformation are indicated by arrows. The position of the membrane relative to the glycoprotein is indicated by the double grey lines. (c) A tomographic slice showing SARS-CoV-2 budding events at ER–Golgi intermediate compartment membranes in VeroE6 cells (EMD-11863) with segmentation of different membrane events at right. The segmentation is adapted from ref. [53] and licensed under CC BY 4.0. To view this license, visit https://creativecommons.org/licenses/by/4.0/.

Figure 5.

Crystal structures still provide the most detailed protein structures. (a) A cartoon representation of the crystal structure of the papain-like protease (PL^pro) from nsp3 of SARS-CoV-2 (PDB 6WZU) with red spheres indicating the residues of the catalytic triad and the prototypic ‘thumb–palm–fingers’ architecture annotated in blue, teal and green. Three different inhibitors that bind to the same pocket are shown in the insets (PDB 7JIR, 7JIT, 7JIW). (b) The crystal structure of the SARS-CoV-2 M^pro (nsp5) homodimer bound to the 11b inhibitor in teal (PDB 6M0 K). The inset shows how density for the inhibitor can be observed in the crystallographic map. (c) Cartoon representations of the SARS-CoV-2 receptor binding domain (RBD; blue) bound to three different ligands. From top to bottom: ACE2 (sea green), EY6A Fab (green), CR3022 Fab (purple).