Abstract
Phase separation is fundamental to cellular organization, yet early events driving this process remain poorly characterized. ATP is highly concentrated in platelet dense granules, where it coexists with divalent cations. Here, we reconstitute ATP phase separation and use nuclear magnetic resonance (NMR) to discover a 1:2 stoichiometric complex between Mg(2+) and ATP. Using a modified NMR pulse sequence of diffusion-ordered spectroscopy (DOSY), we accurately evaluate the size of Mg(2+)-ATP nanoclusters, and identify them as pre-transitional intermediates that grow in hydrodynamic radius at increasing Mg(2+) concentration. Excess Mg(2+) beyond the specific ratio promotes hydration and charge neutralization of the nanoclusters, resulting in the formation of a system-spanning network. The process is characteristic of phase separation coupled to percolation, a mechanism further validated by complementary techniques. Our work reveals how stoichiometric imbalance between interacting components regulates phase transition kinetics and dictates the physical properties of the resulting condensate.