The purified SARS-CoV-2 S glycoprotein ectodomain and human ACE2 PD domain were mixed at a molar ratio of 1:3 and were incubated on ice for 2 hours. The mixture was purified by gel filtration chromatography using a Superose 6 increase 10/300 GL column (GE Healthcare) preequilibrated with 20 mM tris-HCl (pH 7.5), 200 mM NaCl, and 4% glycerol. For cross-linking complex, the buffers of purified SARS-CoV-2 S glycoprotein ectodomain and human ACE2 PD domain were exchanged to 20 mM Hepes (pH 7.5) and 200 mM NaCl; then, SARS-CoV-2 S and human ACE2 were mixed at a molar ratio of 1:3. After incubation on ice for 2 hours, the complex was cross-linked by 0.1% glutaraldehyde, which is commonly used in cryo-EM studies of fragile macromolecular complexes (46, 47). The glutaraldehyde was neutralized by adding 20 mM tris-HCl (pH 7.5) after being incubated on ice for 1 hour. The mixture was run over a Superose 6 increase 10/300 GL column (GE Healthcare) in 20 mM tris-HCl (pH 7.5), 200 mM NaCl, and 4% glycerol. The complex peak fractions were concentrated and assessed by SDS–polyacrylamide gel electrophoresis and negative-staining EM.

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