Cryo-EM sample preparation and data collection
Samples for cryo-EM grid preparation of the 2C06-BG505 DS-SOSIP complex were produced by first mixing 15 μL of purified BG505 SOSIP at 4 mg/mL with 55 μL of 2C06 Fab at 1 mg/mL; n-Dodecyl β-D-maltoside (DDM) was added to have a final concentration of 0.005% (w/v) to prevent preferred orientation and aggregation during vitrification, and the mixture was incubated on ice for 20 min. Cryo-EM grids were prepared by applying 3 μL of sample to a freshly glow discharged carbon-coated copper grid (CF 1.2/1.3 300 mesh). The sample was vitrified in liquid ethane using a Vitrobot Mark IV with a wait time of 30 s, a blot time of 3 s, and a blot force of 0. Cryo-EM data were collected using Leginon softwareFor preparing the 2C09-BG505 DS-SOSIP complexes, Env was mixed with the antibody Fab at 1 to 1.2 M ratio at a final total protein concentration of ~3 mg/mL; DDM (1 mM stock solution) was added to a final concentration of 0.1 mM. 2.7 μL of the mixture was pipetted to a Quantifoil-gold 2/2 holey carbon grids, glow discharged for 30 s in a PELCO easiGlow Glow Discharge Cleaning System prior to cryo-grid preparation, and the grid was blotted for 2.5–4 s and plunge frozen into liquid ethane using a Vitrobot Mark IV (ThermoFisher). Cryo-EM data were collected on a Titan Krios operating at 300 kV, equipped with a K2 Summit detector (Gatan) operating in counting mode. Data were acquired using SerialEM 4.0.The movie frames were aligned and dose-weighted