R1.2 1 3 cu 200 mesh grid

The Bp109-92 and TNFR2-MBP complex was prepared as a 2:3 mixture (5.6 μM Bp109-92 and 8.4 μM TNFR2-MBP) at room temperature for 5 min, and purified in a Superose 6 Increase 10/300 GL column. Three microliters of the complex solution (0.2 mg/mL) were applied to a Quantifoil R1.2/1.3 Cu 200 mesh grid (Quantifoil Micro Tools GmbH, Jena, Germany) that was glow-discharged for 20 s at 20 mA using a JEC-3000FC sputter coater (JEOL). The grid was blotted with a blot force of 0 and a blot time of 3 s in a Vitrobot Mark IV chamber (Thermo Fisher Scientific) equilibrated at 4 °C and 100% humidity, and then immediately plunged into liquid ethane. Excess ethane was removed with filter paper, and the grids were stored in liquid nitrogen. The image dataset was collected using SerialEM^{45 (link)}, yoneoLocr^{46 (link)}, and JEM-Z300FSC (CRYO ARM 300: JEOL) operated at 300 kV with a K3 direct electron detector (Gatan, Pleasanton, CA, USA) in CDS mode. The Ω-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. The nominal magnification was 60,000×. Defocus varied between −0.5 and −2.0 μm. Each movie was fractionated into 60 frames (0.081 s each, total exposure: 4.87 s), with a total dose of 60 e⁻/Ų.

A sample of the J038‐Env‐3BNC117 or J033‐Env‐3BNC117 complexes at 1 mg mL⁻¹ was diluted 1, 0.5, and 0.25 mg mL⁻¹ in PBS buffer. 3 µL of each sample applied to a glow discharged Quantifoil R 1.2/1.3 Cu 200 mesh grid (Glow discharger settings: 0.30 mbar, 15 mAMP, and 5 s). Grids were then plunge frozen in liquid ethane with an FEI vitrobot and stored in liquid nitrogen (Vitrobot settings: 4 °C temperature, 100% humidity, blot time 2 s, blot force 0, wait time 5 s, blot total 1, drain time 0).