To increase the probability of successful crystallization and structure determination, several soluble, cleaved trimers of the SOSIP.664 gp140 design were expressed and purified as described elsewhere (17 (link), 18 (link), 23 (link), 64 (link)). Crystallization candidates based on env genes from HIV-1 isolates KNH1144, ADA, 1182.6.1D2, BG505 and 208.9.C10 were selected from an initial panel of 20, based on their expression and trimer-formation properties, as assessed by SDS-PAGE and BN-PAGE. Notably, for isolates that did not naturally contain residue N332, a point substitution was made to introduce it, thereby creating the 2G12 epitope to facilitate affinity purification. The Env constructs were co-transfected with the furin protease in HEK 293S GnTI−/− cells, which lack N-acetylglucosaminyltransferase I and, therefore, produce glycoproteins bearing only high-mannose (Man5-9) glycans. Secreted SOSIP.664 Env proteins were harvested from supernatants and affinity purified using a 2G12 MAb affinity column. Following a high salt elution process, trimers were purified to size homogeneity using a Superose 6 size exclusion chromatography (SEC) matrix (GE Healthcare).