Room temperature Electron Paramagnetic Resonance (EPR) monitoring of Mn 2+ -SsTroA complex EPR spectroscopy is a useful tool that allows one to probe differences in the metal centers of proteins in solution that may not be apparent in the crystal structures [41] . To further investigate the SsTroA metal center, sample of 100 mM apo-SsTroA was loaded with 100 mM MnCl 2 . The X-band EPR spectra of Mn 2+ -SsTroA at room temperature displays a typical six-line pattern (Fig. 3A) . High-spin Mn 2+ centers typically exhibit EPR resonances with characteristic six-line splitting patterns due to hyperfine interactions between the unpaired electrons and the I = 5/2 55 Mn nucleus, indicating the binding of the Mn 2+ to the protein [42] . Upon entrance of the Mn 2+ into the binding site, the Mn 2+ bound protein reduces the coordination of weakfield water, increasing spin-orbit interactions and zero-field energies relative to that of Mn(H 2 O) 6