In Figure , we show the free energy profile of pore Na + s and pore waters, as well as the time-average z-component of the pore water dipoles (see Methods). Note that our free energy profiles, computed from distribution densities, are designed to find the location and relative affinities of minimumenergy sites. They are not designed to yield quantitative energy barriers as others have done with more advanced enhanced sampling methods. In Figure , the free energy of pore Na + s increases rapidly and stops just before the intracellular gate because the intracellular gate is closed and Na + cannot pass through. We observe three minimum-energy sites for Na + : (i) one centered around the outer-ring of carboxylates (z=-4.3 Å), (ii) one centered around the selectivity filter (z=1.1 Å) and (iii) another centered around the central cavity (z=9.5 Å). There are two corresponding minimum-energy sites for pore waters; one overlapping the first two Na + sites (z=-3.2 Å) and one in the central cavity (z=10.9 Å), slightly offset compared to the Na + one. Note that pore Na + s are coordinated by an average of 4.5 water molecules (Figures and), compared to 5 in the bulk (see Supporting Information). Therefore, pore Na + s remain mostly solvated as they permeate down the channel.