The clade B tier 3 H078.14 UFO-BG trimer was expressed in FreeStyle 293 F cells treated with kifunensine and purified from culture supernatant using a 2G12 affinity column followed by SEC. Fabs PGT124 and 35O22 were transiently transfected into FreeStyle 293F cells (Invitrogen) and purified using a LC-λ capture select column, before further purification by ion exchange chromatography and SEC on a Superdex 200 16/600 column. The trimer complexes were prepared by mixing H078.14 UFO-BG trimer protein with PGT124 and 35O22 at a molar ratio of 1:3.5 for 30 min at room temperature. To decrease glycan heterogeneity, deglycosylation was conducted on the PGT124- and 35O22-bound H078.14 UFO-BG Env protein produced in 293 F cells with Endo H (New England Biolabs) overnight at 4°C. The trimer complexes were subjected to crystal trials after further purification by SEC.

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