Negative-stain sample preparation, data collection, and initial model building

For the negative-stain sample, a volume of 5 μl of SARS-CoV-2 S-ACE2 sample was placed on a plasma-cleaned copper grid for 1 minute. Excess sample on the grid was blotted off using filter paper, and a volume of 5 μl of 0.75% uranyl formate (UF) (Sigma-Aldrich) was added to wash the grid. After blotting, another volume of 5 μl of 0.75% UF was placed on the grid again for 1 minute to stain. Grids were visualized under a Tecnai G2 Spirit 120-kV transmission electron microscope (Thermo Fisher Scientific), and micrographs were taken using an Eagle camera with a nominal magnification of 67,000 ×, yielding a pixel size of 1.74 Å. A total of 41,827 particles were autopicked in EMAN2 (48). After 2D classification, we selected good averages with 13,047 particles for initial model building, which were performed in Relion 3.0 (49).