SOSIP.664 trimers based on the subtype A isolates KNH1144 and BG505 were expressed in 293S GnTI−/− cells and purified by 2G12 affinity followed by size exclusion chromatography (Superdex S200, GE Healthcare) in PBS (20 mM sodium phosphate pH 7.4, 150 mM NaCl with 1 mM EDTA and 0.02 % sodium azide) as described previously 19 (link), 63 (link). Soluble two-domain CD4 (sCD4) 64 (link), HIV-IG and monoclonal antibodies 17b 4 (link), 14 (link), 23 (link), 24 (link), b12 20 (link), 65 (link), 66 (link), 2G12 27 (link), 67 (link), and VRC01 68 (link) were obtained from the NIH AIDS reagents program. Fabs were prepared using Pierce Fab preparation kit (Thermo Scientific) according to manufacturer’s instructions. PGV04, PGT123, and PG9 Fabs were prepared as described previously 69 (link). All proteins were buffer exchanged into PBS. Complexes with SOSIP.664 trimers were formed by overnight incubation with a threefold molar excess of ligand (relative to each protomer) at 4°C, except for the 17b Fab complex, which was incubated for 72 h at room temperature. SDS- and BN-PAGE analyses were performed with the same samples used for HDX, to ensure that complexes had formed and that no degradation had occurred throughout the incubations (Supplementary Figure 3).