To prepare the cryo-EM sample of SARS-CoV-2 S trimer, the sample was diluted into around 2 mg/ml using buffer with 20 mM tris-HCl (pH 7.5), 200 mM NaCl, and 4% glycerol. A 2.2-μl aliquot of the S trimer sample was applied on a plasma-cleaned holey carbon grid (R2/1, 200 mesh; Quantifoil) or Graphene Oxide-Lacey Carbon grid (300 mesh, EM Resolutions). The grid was blotted with Vitrobot Mark IV (Thermo Fisher Scientific) using a blot force of −1 and 1-s blot time at 100% humidity and 8°C and then plunged into liquid ethane cooled by liquid nitrogen. To prepare the cryo-EM sample of S-ACE2 complex with or without cross-linking, we used Graphene Oxide-Lacey Carbon grid (300 mesh, EMR) and adopted the same vitrification procedure as for the S trimer.

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