2.2 Crystallization
All ppnP proteins were crystallized using the sitting drop vapor diffusion method by mixing 0.2μl of protein and 0.2μl of reservoir solution at 20 ℃.
1) The crystal of EcppnP suitable for x-ray diffraction was grown in a reservoir solution consisting of 2.0 M Ammonium sulfate (dibasic), 0.1M CAPS/Sodium hydroxide pH 10.5, 0.2M Lithium sulfate (RIGAKU).
2) Selenomethionine-derived EcppnP suitable for x-ray diffraction was grown in reservoir solution consisting of 1.26 M Ammonium sulfate (dibasic), 0.1M Sodium cacodylate/Hydrochloric acid pH 6.5 (RIGAKU).
3) The crystal of StppnP suitable for x-ray diffraction was grown in reservoir solution consisting of 0.2 M Lithium sulfate monohydrate, 0.1 M Tris pH 8.5, 25% w/v Polyethylene glycol 3,350 (Hampton Research).
4) The crystal of VcppnP suitable for x-ray diffraction was grown in a reservoir solution consisting of 0.2 M Ammonium sulfate, 30% w/v Polyethylene glycol 8,000 (Hampton Research).
5) The crystal of PappnP suitable for x-ray diffraction was grown in a reservoir solution consisting of 0.1 M BIS-TRIS pH 5.5, 2.0 M Ammonium sulfate. (Hampton Research)