Clearly defined intra-organelle and cytosolic spaces are inherent to the biological concept of the fusion pore (figs. 1a and 1b). Therefore, mutually exclusive groups of intra-organelle and cytosolic water beads becomes a convenient definition for our simulation systems (figs. 1c to 1e). Then, collective variable ξ e dynamically selects at each molecular dynamics step if a water bead is intra-organelle or cytosolic. Specifically, a water molecule belongs to the intraorganelle subgroup if it fulfills 1 out of the following 3 conditions: (i) W is above the Z plane that contains the geometric center of the upper bilayer, or (ii) W is below the Z plane that contains the geometric center of the lower bilayer, or (iii) W is inside the cylinder of radius R max that defines expansion. Any W bead not belonging to the intra-organelle subgroup is considered cytosolic. This is not only an improvement in terms of computational performance but also, it avoids possible errors due to water beads permeating through the bilayers from the intra-organelle space into the cytosol and vice-versa (especially for very long µs-length simulations).