Calcium induced CHGB aggregation and single particle analysis of CHGB dimers. Calcium induced CHGB aggregation and single particle analysis of CHGB dimers. (A) Typical SEC profile of partially purified CHGB in a Superose 6 column. The dimer peak (U2) was collected for further preparation. The tetramer peak (U4) could be separated although it was not as stable as the dimer. (B) Calcium-induced aggregation. 20 μg CHGB incubated with different [CaCl2] for 30 min at RT and OD was measured at 320 nm. Change in absorbance (ΔAbs @320 nm) was plotted against log [CaCl2] and fitted with a Hill equation as follows:ΔAbs=ΔAbsmax1+10{log(kD)-log[CaCl2]}×n Fitting (black line) yielded kd ∼ 0.25 mM, a Hill coefficient n ∼2.0, suggesting the apparent positive cooperativity in the Ca2+-stimulated aggregation. Error bars represent SD (n = 3). (C–F) EM images of negatively stained (2% PTA) CHGB incubated with different [CaCl2]. Red circles highlight some of the CHGB dimers (C) or aggregates (D–F). Scale bars: 25 nm. (G) Images of negatively stained CHGB dimers in detergents. The protein concentration was 0.1 mg/ml. The stain was 2.0% PTA/KOH pH 8.0. Scale bar: 100 nm. (H) A 30-Å negative-stain map calculated from 3,200 particles. Scale bar: 2.0 nm. (I) Typical cryoEM images of the CHGB dimers on the surface of a ChemiC-Ni-NTA grid. Some of the CHGB particles are highlighted by red circles. The protein is in black in the raw images. Scale bar: 20 nm. Gaya P Yadav et al. LSA 2018;1:e201800139 © 2018 Yadav et al.