R2 2 300 mesh grids

BNM-III-170-BG505-17b and M48U1-BG505-17b complexes were assembled by incubating CD4m compounds BNM-III-170 or M48U1 with BG505 overnight at room temperature at a molar ratio of 10:1 (CD4m:trimer). 17b Fab was added the next day at a 9:1 ratio (Fab:trimer) and incubated at room temperature for 2–4 h. Complexes were purified by SEC on a Superdex 200 Increase GL 50/150 or a Superdex 200 Increase 10/300 GL column (GE Healthcare) and fractions containing CD4m-BG505-17b complexes were concentrated to 1.4-1.5 mg/mL. Cryo-EM grids were frozen using a Mark IV Vitrobot (ThermoFisher) at 22 ^oC and 100% humidity. 3.1 µL of the sample was applied to Quantifoil R2/2 300 mesh grids, blotted for 3 or 3.5 s, and plunge frozen into liquid ethane. Grids were then transferred to grid boxes in liquid nitrogen and stored until data collection.

Samples of late log-phase liquid cultures (Table 1) were placed on glow-discharged Quantifoil R2/2 300 Mesh grids and were vitrified by plunge freezing in liquid ethane by using a Mark IV Vitribot (FEI, Hillsboro, OR). Samples were imaged with a Tecnai G2 F30 300 kV scanning TEM (FEI) fitted with a K2 Summit 14.2 Megapixel direct detection camera (Gatan, Pleasenton, CA) and a Gatan K2 direct electron detector. Images were processed with digital micrograph software (Gatan) as well as SerialEM (Univ. Colorado, Boulder). The Cryo-EM images presented in this study are deposited in the EMBL-EBI Biostudies database (https://www.ebi.ac.uk/biostudies) under accession S-BSST1223.

ATP synthase elution fractions from the gel filtration run were of a concentration of ~10 mg/mL and aliquots were diluted in buffer D to 0.75 mg/mL immediately before application onto grids. A home-made amorphous carbon layer ~3 nm thick was floated onto Quantifoil R2/2 300-mesh grids and grids were glow-discharged immediately before sample application. A sample volume of 3 µL was applied onto grids and vitrified by plunge-freezing into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV. A wait time of 30 s was used with a blot time of 3 s. Micrograph data was collected using EPU 1.9 on a Titan Krios (ThermoFisher Scientific) operated at 300 kV at a nominal magnification of 165 kx (0.83 Å/pixel) with a Quantum K2 camera (Gatan) using a slit width of 20 eV. With an objective lens aperture of 70 µm, images were collected with an exposure rate of 4.26 electrons/pixel/second with 5 s exposure fractionated into 20 frames. A total of 15439 movies were collected.