To generate the apo-SsTroA protein, SsTroA protein was treated as described previously with minor modification [31] . Briefly, the purified protein was dialyzed against 20 mM sodium acetate buffer (pH 6.5) and 20 mM EDTA for 3 days. The protein was then dialyzed against EDTA-free buffers for 3 days. All dialysis steps were performed at 4uC with stirring, and the buffer was replaced every 12 hours. Removal of metal was confirmed by ICP-MS (Inductively coupled plasma mass spectrometry). Clear plastic tips and containers were employed to prevent metal crosscontamination. All plasticwares were treated for .2 hours with 0.2 M EDTA to remove the contaminating metals, and all buffer solutions were passed through chelating Sepharose resin and stored in plastic beakers.