Membrane insertion of CHGB introduces positive curvature in bilayers. Membrane insertion of CHGB introduces positive curvature in bilayers. Purified CHGB protein was reconstituted in lipid vesicles made of egg PC. Empty vesicles were prepared in the same way without CHGB. The vesicles were diluted by fivefold in a buffer containing 20 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1.0 mM EDTA, and 2.0 mM β-ME before being loaded on carbon-coated grids and stained with 2.0% PTA (phosphotungstic acid/KOH, pH 8.0). Typical images of empty vesicles (A), CHGB reconstituted vesicles at 1:10 protein/lipid weight ratio (1:1,000 in molar PLR or ∼40 CHGB dimers per 100 nm vesicle) (B), CHGB reconstituted vesicles at 1:5 protein/lipid weight ratio (1:500 in molar PLR or ∼80 CHGB dimers per 100 nm vesicle) (C), and CHGB-reconstituted vesicles at 1:1 protein/lipid weight ratio (1:100 in molar PLR) (D) are shown. The experiments were repeated more than three times with very similar results. Budding compartments with positive curvature are marked with red arrowheads in (B–D). In (B), the nanospheres are marked at the tops of the nanotubules. Scale bars in A–D are all 25 nm. (E) 10 μg CHGB in egg PC vesicles treated with trypsin at RT. PMSF was used to stop the reaction. Samples collected at various time points were separated in a 4–20% SDS–PAGE gel. The stable short fragment at ∼20 kD was collected for mass spectrometry analysis (marked as MIF), which is named as CHGB-MIF in Fig 3D–F. (F) Model for insertion of CHGB in membranes to cause positive curvature. Gaya P Yadav et al. LSA 2018;1:e201800139 © 2018 Yadav et al.