Hiload 16 90 superdex 200

54-4H03 Fab or 54-1G05 Fab was incubated with the purified HA trimer in a molar ratio of 4.5:1 overnight at 4°C. The Fab-HA complexes were purified by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare) in 10 mM Tris pH 8.0, 50 mM NaCl, and 0.02% NaN₃, and concentrated to 10 mg mL^-1.

2G12 Fab was generated from IgG with IdeS (Genovis) digestion in buffer containing 100 mM Bis-Tris pH 6.5, 150 mM NaCl. The reaction was incubated at 37°C for 2 hours followed by SEC by Hiload 16/90 Superdex 200 column (GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl. To form immune complexes, HA and 2G12, as a domain-swapped Fab dimer, were incubated at a 1:3 ratio of HA to 2G12 for 2 hours at room temperature. To prepare negative-stain EM grids, immune complexes at ~30 μg/ml were deposited onto glow-discharged carbon-coated 400 mesh copper grids (Electron Microscopy Sciences, EMS) and stained with 2% w/v uranyl formate. Grids were imaged on a 200kV Tecnai T20 transmission electron microscope (FEI) with an Eagle CCD 4k camera (FEI) at 62,000x nominal magnification. Micrographs were collected using Leginon [82 (link)], particles were picked and extracted using Appion [83 (link)], and particles were categorized into reference-free 2D class averages using Relion [84 (link),85 (link)]. We were unable, however, to get convergence to achieve higher resolution 3D reconstructions. The putative binding interactions between 2G12 Fab and N165 and N246 of wild type Bris07 HA, N246 of Bris07 N165A HA were fitted based on the negative-stain EM and the binding features of 2G12 to oligomannose identified in a previous HIV-1 structural study [11 (link)].