To understand the molecular mechanism of SNARE induced membrane fusion, an understanding of the structure of the SNARE complex was essential. Using truncated soluble t- and v-SNAREs, the structure of the resultant complex formed, termed the core domain, was determined at 2.4 Å using x-ray crystallography
. As it became clear from subsequent studies , the x-ray crystallographic study  nuanced the importance of membrane-associated SNAREs. As with most membrane proteins, the interaction and resultant arrangement of the t-/v- SNARE complex were found to be quite different when t-/v-SNAREs are in solution as opposed in association with membrane . Using atomic force microscopy and bilayer electrophysiological assays, Bhanu P. Jena and his research team demonstrated for the first time that full length t-SNARE and v-SNARE in opposing bilayers interact in a circular array (forming ring-like channels), to form conducting channels in the presence of calcium [7, 8]. On the contrary, when the same full length v- and t-SNAREs are in solution (in absence of membrane), or even when one of the SNAREs is in solution, t-/v-SNARE interactions fail to form such conducting channels . These studies by Jena [7, 8] were a major breakthrough in our understanding of SNARE-induced membrane fusion. They demonstrated that t- SNAREs and v-SNARE should reside in opposing membranes to allow appropriate t-/v-SNARE interactions, leading to membrane fusion in the presence of calcium. They further prove that in the physiological state in cells, both SNAREs and Ca2+ operate as the minimal fusion machinery . Using SNAREreconstituted liposomes and bilayers , Jena showed that (i) there is a low fusion rate (τ=16 min) between t- and v-SNARE-reconstituted liposomes in the absence of Ca2+; however, (ii) exposure of t-/v-SNARE liposomes to Ca2+, drives vesicle fusion on a near physiological relevant time-scale (τ ~10s), establishing an essential role of Ca2+ in membrane fusion. Since the Ca2+ effect on membrane fusion in SNARE-reconstituted liposomes was found to be downstream of SNAREs, it suggests a regulatory role for Ca2+-binding proteins in membrane fusion in
the physiological state in cells . These studies clearly reveal that not just SNAREs, but both SNAREs and Ca2+ are the minimal fusion machinery in cells [7, 8]. Native and synthetic vesicles exhibit a significant negative surface charge primarily due to the polar phosphate head groups. These polar head groups produce a repulsive force, preventing aggregation and fusion of apposing vesicles. In their pioneering studies , Jena and his research team further demonstrate that SNAREs bring opposing bilayers closer to within a distance of 2–3 Å, thus allowing Ca2+ to interact with the phospholipid head groups, and bridging them. The bound Ca2+ then leads to the exclusion of water between the bilayers at the bridging site, allowing lipid mixing and membrane fusion. Hence SNAREs, besides bringing opposing bilayers closer, dictate the site and size of the fusion area during cell secretion . The size of the t-/v-SNARE complex forming the pore is dictated by the curvature of the opposing membranes, hence depends on the size of t-/v-SNARE-reconstituted vesicles. The smaller the vesicles, the smaller the pores formed . The assembly and disassembly of the SNARE complex, has also been determined by Jena and his research team .