Negative staining electron microscopy analysis shows that Syt1C2AB forms ring-like structures in buffer solution containing soluble PIP2 (PIP2-diC4) and 1 mM free Mg2+ (A), and these rings are very similar to those observed on monolayers containing 40% DOPS (B). (C) The average diameter of the Syt1C2AB soluble ring-oligomers are slightly but not significantly larger (32 ± 3 nm) compared to the rings (28 ± 2 nm) formed on lipid monolayer. These oligomeric rings were not unique to PIP2-diC4 and other polyvalent anions, like IP3 (D) and ATP (E) also induced the oligomerization of Syt1C2AB. The prevalence and stability of the Syt1C2AB were similar for all the polyvalent anions tested (F). The scale bar represents 100 nm in the main figure and 50 nm for the insets (Panels A, B, D, E). Average values and standard deviations from a minimum of 3 independent experiments are shown.