Fig. 4.

Cryo-EM analysis of liposome clustering by lnC₂AB. Samples containing 3 μM or 10 μM lnC₂AB, 1 mM Ca²⁺, and phospholipid vesicles (0.3 mg/mL lipids) were incubated and fast-frozen on EM grids (5 min after mixing the reagents). (A) Examples of the cryo-EM images obtained. Arrows indicate liposome pairs with apparent intermembrane distances of 4 nm or less. (Scale bar: 100 nm.) (B) Overall distribution of apparent distances measured between 697 selected liposome pairs bridged by lnC₂AB. (C and D) Distribution of apparent distances measured between liposome pairs in samples containing 3 μM lnC₂AB, yielding an lnC₂AB-to-lipid ratio of 1:120 (C), or 10 μM lnC₂AB, yielding an lnC₂AB-to-lipid ratio of 1:36 (D). The distribution of B includes all of the distances included in C and D. Note that protein-free liposomes do not cluster under the conditions of our experiments (20).