Quantum k3 direct electron detector

The SEC peak fraction containing the purified 4F2hc-LAT2::D11vs complex (Fig. 1a) was concentrated to ~ 3 mg/ml using an Amicon 100-kDa molecular weight cut-off device and centrifuged (20,000 × g, 4 °C, 5 min). A protein sample of 3 µL was applied to a R2/1 200 mesh copper holey-carbon grid (Quantifoil, Germany) that has been glow discharged for 25 s at 10 mA and 0.25 mbar. Samples were blotted for 3 s and vitrified in liquid ethane using a Vitrobot Mark IV system (ThermoFisher) at 4 °C and 100% humidity. Data were acquired on a Titan Krios G3 cryo-TEM (ThermoFisher) operated at 300 kV and equipped with a Quantum-K3 direct electron detector (Gatan). A total of 20,512 movies were collected in counting mode at a defocus range of -0.8 to -1.8 μm and a magnification of 105,000x, which corresponds to a calibrated pixel size of 0.822 Å. Movies were recorded for 1.491 s with a dose rate of 1.255 e⁻/Ų/frame, resulting in a total accumulated dose on the specimen level of approximately 50.2 e⁻/Ų per exposure (Supplementary Table S1).

Purified GPR (2.5 μL at 3.5 mg/mL) was applied to Quantifoil R1.2/1.3 300 mesh copper holey-carbon grids that were glow discharged for 30 s at 10 mA and 0.25 mbar. Samples were blotted for 5 s and vitrified in liquid ethane using a Vitrobot Mark IV (ThermoFisher) at 4 °C and 100% humidity. Data were acquired using SerialEM^{48 (link)} on a Titan Krios G3 (ThermoFisher) operated at 300 kV and equipped with a Quantum-K3 direct electron detector (Gatan). Micrograph movies were recorded in counting mode at a magnification of ×130,000 (corresponding to a calibrated pixel size of 0.645 Å) and a defocus range of −0.9 to −1.9 μm. A total of 20,307 movies were collected, each comprising 40 frames, with a total dose of 54.23 e⁻/Ų.