








key: cord-356207-tpn5cg4n
authors: Beniac, Daniel R; Andonov, Anton; Grudeski, Elsie; Booth, Tim F
title: Architecture of the SARS coronavirus prefusion spike
date: 2006-07-16
journal: Nat Struct Mol Biol
DOI: 10.1038/nsmb1123
sha: 
doc_id: 356207
cord_uid: tpn5cg4n

The emergence in 2003 of a new coronavirus (CoV) responsible for the atypical pneumonia termed severe acute respiratory syndrome (SARS) was a stark reminder that hitherto unknown viruses have the potential to cross species barriers to become new human pathogens. Here we describe the SARS-CoV 'spike' structure determined by single-particle cryo-EM, along with the docked atomic structures of the receptor-binding domain and prefusion core. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nsmb1123) contains supplementary material, which is available to authorized users.

and dialysed against PBS. SARS-CoV-enriched fractions were checked by SDS-Page and Western blotting, and rendered non-infectious by irradiation in a gamma cell on dry ice with a 2 Mrad exposure for 90 minutes. The dose was chosen as sufficient for viral inactivation 1 , while retaining antibody 1,2 or enzyme functional activities 3 . Previous studies have indicated that viral RNA is highly sensitive to radiation and that viruses were by 2 Mrad 1 , even when cooled to minimize heat damage to proteins, whereas antigenicity of viral proteins and antibody binding titres were maintained after doses of 3.6 Mrad 1 or 5 Mrad 3 . Irradiated specimens were tested for infectivity by inoculation onto Vero E6 cells, and examined for cytopathogenic effects for 10 days, followed by blind passage of the cells and testing for the growth of SARS-CoV by PCR 4 .

Cryo-Electron microscopy. Virus samples (4µl) were applied to glow-discharged holey carbon films supported on 400-mesh copper grids. After blotting immediately for 2-5 seconds with filter paper, grids were plunged into liquid ethane cooled by liquid nitrogen, using a custom built gravity-operated freezing device. Specimens were transferred to a Tecnai 20 G2 transmission electron microscope (FEI) operated at 200kV, equipped with a Gatan 626.DH low-temperature specimen holder.

Observations were made at temperatures of ~ -185°C and images recorded at 29,000X magnification on Kodak SO -163 electron image film at a dose of 10-20 electrons/Å 2 with an exposure of 1-2 seconds.

Film was developed in Kodak D19 for 12 minutes at room temperature. Immuno-gold stained samples were imaged at room temperature in the Tecnai 20, and digital images were collected using either a Gatan MSC or AMT Advantage XR-12 digital cameras.

Image processing. The exact magnification in the microscope was determined to be 29,968X using a calibration grid ( 

Formvar-carbon coated 400-mesh nickel grids were floated on drops of purified SARS-CoV (40μl) for 1 minute. All incubations were carried out at 20 °C. Grids were then washed in PBS, followed by a 10-minute block in PBS-G-BSA (PBS pH7.2, 0.2% glycine, 2% BSA). After washing in PBS-G (PBS pH7.2, 0.2% glycine), grids were then incubated in primary antibody diluted in PBS-G-BSA or convalescent patient serum diluted in PBS, followed by washes in PBS-G and then incubated in secondary antibody (conjugated to 5 or 10 nm gold), followed again by washing in PBS-G. Grids were fixed (1% paraformaldehyde, 1% glutaraldehyde in PBS), washed in deionised water and negatively stained in either 2% uranyl acetate or 2% methylamine tungstate (Nanoprobes, Yaphank, NY.). In the case of nucleocapsid labelling, specimens were permeabilised by pre-treatment with 0.05% NP-40 for in PBS on ice. In all experiments negative controls were run which included omission of the primary antibody to test for non-specific binding.

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