R2.0 2.0 cu 300 mesh grid

For the preparation of a complex sample for cryo-EM, BA.2.75 S ectodomain solution was incubated at 37°C for 1 hour before use. Purified ACE2 was incubated with BA.2.75 S ectodomain at a molar ratio of 1:3.2 at 18°C for 1 hour. A 0.1% (w/v) n-octyl-β-D-glucoside solution (Dojindo, Cat# O001) was added to BA.2.75 S ectodomain solution with or without ACE2 to a final concentration of 0.01%. The sample was then applied to a Quantifoil R2.0/2.0 Cu 300 mesh grid (Quantifoil Micro Tools GmbH), which had been freshly glow-discharged for 120 seconds at 10 mA using PIB-10 (Vacuum Device). The samples were plunged into liquid ethane using a Vitrobot mark IV (Thermo Fisher Scientific) with the following settings: temperature 18°C, humidity 100%, blotting time 5 seconds, and blotting force 5. Micrographs were collected on a Krios G4 system (Thermo Fisher Scientific) operated at 300 kV with a K3 direct electron detector (Gatan) at a nominal magnification of 130,000 (0.67 per physical pixel) using a GIF-Biocontinuum energy filter (Gatan) with a 20-eV slit width. Each micrograph was collected with a total exposure of 1.5 s and a total dose of 50 e/Å2 over 50 frames. A total of 3308 and 3248 micrographs of S and S-ACE2 complexes, respectively, were collected. All micrographs were collected at a nominal defocus range of 0.8–1.8 μm using EPU software v2.14 (Thermo Fisher Scientific).