Reconstitutions with Zn 2+ and Mn 2+ were performed as described previously [31] with minor modifications. Briefly, 10 monomer equivalents of Zn 2+ or Mn 2+ were added to apo-SsTroA protein at 4uC for 12 hours. The samples, were then subjected to three rounds of concentration with Amicon Ultra 15 centrifugal filters (Millipore) followed by dilution to remove unbound metal. The metal content of recombinant SsTroA samples exchanged into metal-free 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl, reconstituted SsTroA samples, and buffer controls were determined. Metal ion analysis was determined by ICP-MS, at the Analysis Center of Tsinghua University (Beijing, China). Metal content was analyzed on an XSeries II (Thermo-Fisher) apparatus. Each sample was quantified three times and averaged.