R1.2 1 3 300 mesh au holey carbon grids

Freshly purified 2.5–3.5 mg/ml V-ATPase in buffer C was applied to Quantifoil R1.2/1.3 300 mesh Au holey carbon grids (Quantifoil). The grids were then blotted and plunged into liquid ethane for flash freezing using a Vitrobot Mark IV (FEI). The grids were imaged in a 200 keV Talos Arctica (FEI) with a Gatan K3 Summit direct electron detector (Gatan) in super-resolution mode using the data-collection software Serial EM. Data were collected at 0.735 Å/pixel with a counted rate of 42 electrons per physical pixel per second. Images were recorded for 4 s exposures in 50 subframes with a total dose of 80 electrons per Ų and a defocus range of −1.0 to −2.0 μm.

Prior to freezing grids, the OX2R-mG_sqiN-scFv16 complex was concentrated to 9.5 mg/ml. Cryo-EM grids were prepared by applying 3.5 μl of this sample to glow-discharged Quantifoil R1.2/1.3 300-mesh Au holey carbon grids (Quantifoil Micro Tools GmbH, Germany), blotted for 4.5 s under 100% humidity at 4 °C and plunge frozen in liquid ethane cooled by liquid nitrogen using a Mark IV Vitrobot. SerialEM software was used for automated data collection. Images were recorded on a Titan Krios microscope (FEI) operated at 300 kV with a K3 direct electron detector (Gatan) in super-resolution correlated-double sampling counting mode using a slit width of 20 eV on a GIF-Quantum energy filter (Supplementary Fig. 12a). Images were recorded at a nominal magnification of ×81,000, corresponding to a pixel size of 1.08 Å, and a target defocus range from −1.6 to −2.6 μm. Each movie stack was dose-fractionated over 32 frames for a total of 9 s under a dose rate of 8 e⁻/pixel/sec, resulting in a total dose of ~ 64 e⁻/ Ų.