Fig. 4: Structural and biochemical analyses of the gC1q/eCUB interaction.

a, b Cryo-EM density map (a) and atomic model (b) of the C1ql1_gC1q hexamer and BAI3_eCUB complex. In this complex structure, the C1ql1_gC1q hexamer interacts with four molecules of BAI3_eCUB. A rotational view depicting the interaction of each C1ql1_gC1q trimer and two BAI3_eCUB molecules is illustrated, further showcasing their molecular interfaces. The bound Ca²⁺ in the central axis and the interface, and the Ca²⁺-axis traversing the central calcium ions in each trimer are indicated by gray spheres and blue lines, respectively. c Cryo-EM structure of the focused subcomplex containing one gC1q trimer and one molecule of eCUB. BAI3_eCUB contains a classical CUB domain and a C-terminal CUB extension. d Detailed interface between C1ql1_gC1q and BAI3_eCUB. The interface Ca²⁺ ion was represented by a gray sphere. e Density-fit-model mode to show the key interface residues and the interface Ca²⁺ ion. f Overall structure of BAI3_eCUB. The CUB domain contains eight β-strands, while the CUB extension contains an α-helix (α^C) followed by a loop region (L^C). The atomic model of residues 28–203 in eCUB can be built, while the others in the two termini are flexible, indicated by dashed lines. Two and three disulfide bonds are observed in the CUB domain and CUB-extension regions, respectively. g, h ITC-based binding analyses between C1ql1_gC1q variants in their trimeric forms and BAI3_eCUB (g) and between C1ql1_gC1q trimer and BAI3_eCUB variants (h). i Multisequence alignment of the main gC1q-interacting region in the BAI family members. The key interface residues are labeled by arrowheads. The two interface residues that are not conserved in BAI1 are highlighted by blue circles. j aSEC-based analysis showing the disruptive interaction between C1ql1_gC1q and the E60R/T65R mutant of BAI3_eCUB.