Figure S2. 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:

$$\Delta \text{Abs}=\frac{\Delta {\text{Abs}}_{\text{max}}}{1+{10}^{\left\{log\right(k_D)-log[CaC{l}_2\left]\right\}\times n}}$$

Fitting (black line) yielded k_d ∼ 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.