Endo h treatment

For crystallography, a minimally glycosylated eOD-GT8 [His6x-tagged eOD-GT8minglyc (16 (link))] was produced by transiently transfected 293S (GnTI^−/−) cells and then purified using a HisTrap nickel column (GE Healthcare), followed by size exclusion chromatography using a Superdex75 column (GE Healthcare). The Fab constructs of the naive Abs were first affinity purified with human lambda resin (Life Technologies), followed by cation exchange chromatography (GE Healthcare). After incubating the purified Fab with a molar excess of purified eOD-GT8minglyc, the complex was deglycosylated by an Endo H treatment (New England Biolabs), followed by purification over Superdex200 size exclusion chromatography (GE Healthcare). The deglycosylated complex entered crystallization trials using an automated CrystalMation robotic system (Rigaku) at concentrations between 5 and 15 mg/ml. Crystals of the complex of the IOMA-class naive B cell CLK31 Fab with eOD-GT8minglyc (PDB ID: 6D2P; 2.60 Å resolution) were obtained at 20°C in a solution at pH 5.0 of 20% w/v PEG6000, and 0.1 M citric acid (pH 4.0). Crystals were cryoprotected in 25% glycerol followed by fast plunging in liquid nitrogen.