X-band EPR measurement was performed on an ER 200D SRC spectrometer (Bruker Instruments) equipped with a rectangular microwave cavity, operating at a microwave frequency of 9.53 GHz at 25uC. Three scans were collected at a microwave power of 20 mW. The center field of the EPR scans was set to 3,500 G with a sweep width of 1,000 G. EPR samples were prepared by titrating Mn 2+ into a solution of apo-SsTroA. Stock solutions of MnCl 2 were diluted in protein buffer with doubly distilled water. To monitor Mn 2+ binding, one syringe was loaded with a Mn 2+ -saturated SsTroA solution (100 mM SsTroA and 100 mM MnCl 2 ) in protein buffer and the other was loaded with 100 mM ZnSO 4 freshly prepared in the same buffer. After each addition the sample was incubated for 10 minutes at 4uC prior to recording the EPR spectrum.