Cu400 em grids

The nanoparticles produced in Expi293 cells were purified using agarose-bound lectin beads (Agarose Galanthus Nivalis Lectin, Vector Laboratories) followed by size-exclusion chromatography (GE Healthcare) using the Superose 6 Increase 10/300 GL Column. The proteins were further dialyzed into Tris-buffered saline (TBS). A total of 3 μL of purified proteins was adsorbed onto glow-discharged carbon-coated Cu400 EM grids (Electron Microscopy Sciences). The grids were then stained with 3 μL of 2% uranyl acetate, blotted, and stained again with 3 μL of the stain followed by a final blot. Image collection and data processing was performed on a FEI Tecnai T12 microscope equipped with a Oneview Gatan camera at 90,450X magnification at the camera and a pixel size of 1.66 Å.

SOSIP/Fab complexes were made by mixing 15 μg AMC018 SOSIPv4.2 with 6-fold per protomer molar excess Fab and allowed to incubate for 18 to 24 hr at room temperature. Complex samples were SEC purified using a SuperoseTM 6 Increase 10/300 GL (GE Healthcare) column to remove excess Fab prior to EM grid preparation. Fractions containing the SOSIP/Fab complexes were pooled and concentrated using 10 kDa Amicon® spin concentrators (Millipore). Samples were diluted to 0.03 mg/mL in TBS (0.05 M Tris pH 7.4, 0.15 M NaCl) and adsorbed onto glow discharged carbon-coated Cu400 EM grids (Electron Microscopy Sciences) and blotted after 10 s. The grids were then stained with 3 μL of 2% (w/v) uranyl formate, immediately blotted, and stained again for 45 s followed by a final blot. Image collection and data processing was performed as described previously on a FEI Talos microscope (1.98 Å/pixel; 72,000 × magnification) with an electron dose of ~25 electrons/Ų using Leginon (Pugach et al., 2015 (link); Suloway et al., 2005 (link)). 2D classification, 3D sorting and 3D refinement conducted using Relion v3.0 (Zivanov et al., 2018 (link)). EM density maps were visualized using UCSF Chimera (Pettersen et al., 2004 (link)).