Cryo-EM grid preparation and data collection

For cryo-EM grids preparation of the CB₂R-G_i complexes, 3 μL of the concentrated protein was loaded to a glow-discharged holey carbon grid (CryoMatrix Amorphous alloy film R1.2/1.3, 300 mesh), and subsequently were plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The chamber of Vitrobot was set to 100% humidity at 4 °C. The sample was blotted for 2.5 s with blot force 2. Cryo-EM images were collected on a Titan Krios microscope operated at 300 kV equipped with a Gatan Quantum energy filter, with a slit width of 20 eV, a Gatan K2 summit direct electron camera (Gatan). Images were taken at a dose rate of 8e⁻/Ų/s with a defocus range of −0.8 to −2.0 μm using SerialEM software⁶⁴ in EFTEM nanoprobe mode, with 50 μm C2 aperture, at a calibrated magnification of 130,000 corresponding to a magnified pixel size of 1.04 Å. The total exposure time was 8.1 s and 45 frames were recorded per micrograph.