Crystallization and Structure Determination of Pyrophosphatase

Hampton Research packs (PEGRx1, PEGRx2, Index, Crystal Screen, and Crystal Screen 2) were used for the initial crystallization screen (sitting-drop vapor diffusion method). To obtain a crystal suitable for X-ray diffraction, we optimized crystallization by using the initial three conditions. Larger crystals were obtained from drops that contained 1 μL protein and 1 μL solution from the well containing 2.2 M DL-malic acid, pH 6.5, with or without 5 mM PPi (hanging-drop method). The crystallization conditions of the mutants are similar to the wild-type. Prior to X-ray data collection, crystals were soaked for approximately 1 min in the reservoir solution supplemented with 20% (v/v) glycerol as a cryoprotectant, and then flash cooled in liquid nitrogen.
Data sets were collected at 100 K at the Shanghai Synchrotron Radiation Facility (Shanghai, China). Data sets were indexed and integrated using XDS [55 (link)] and scaled using Aimless from the CCP4 package [56 (link)]. Structures were determined by Phaser [57 (link)] with a molecular replacement method using the structure of PPase (PDB: 1i6t) as the search model. Structure refinement and water updating were performed using Phenix refine [58 (link)] and Coot [59 (link)] manual adjustment. The final structure validations were performed using MolProbity [60 (link)]. Figures for all structures were generated using PyMOL [61 ] or Coot.