Detection of bound Na + in a membrane protein by pulse EPR

Na + ions generate electron density signals similar to water, making their detection in membrane protein crystal structures challenging. Combined electron spin echo envelope modulation (ESEEM) spectroscopy with site‐directed spin labeling revealed 23 Na signals at two labeled sites in the Na + /aspartate symporter Glt Tk . Comparison of experimental 23 Na nuclear quadrupole parameters with density functional theory calculations required a single, physically motivated torsional adjustment to improve Na + coordination. The nitroxide–Na + distances derived from molecular dynamics simulations and the rotamer analysis of the spin labels, based on a crystal structure, aligned with the experimental values within an uncertainty of 1.5 Å. We conclude that ESEEM is an effective method for localizing and studying the coordination of protein‐bound Na + ions.