Pegrx 2

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.

Cryo-EM samples were prepared on Quantifoil R1.2/1.3 mesh 200 Cu grids, Quantifoil R2/1 mesh 200 Cu grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany), and C-flat-2/1 mesh 200 Cu grids (Protochips, Morrisville, NC, USA) using Q150T ES glow discharge system (Quorum Technologies, East Sussex, England) and Vitrobot Mark IV (Thermo Fisher Scientific, MA, USA). Protein crystals were produced using the following crystallization kits: Crystal Screen (Hampton Research, HR2-110), PEGRx 2 (Hampton Research, HR2-084) and crystallization robots: Phoenix (Art Robbins Instruments, Sunnyvale, CA, USA), Oryx (Douglas Instruments, Hungerford, UK). For biochemical experiments the following fluorescently labeled deoxynucleotides were used: Fluorescein-dUTP (Thermo Scientifc, R0101), Texas Red-dCTP (Jena Bioscience, NU-809-TXR-S). The results of activity assays were visualized by fluorescence readout using Amersham Typhoon (Cytiva, Marlborough, MA, USA) biomolecular imager.

Crystallization screening of the N-PAS domain was performed using the sitting-drop vapour-diffusion method. Up to 14 different series of screening solutions were prepared, including Index, Index 2, Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, SaltRx, SaltRx 2, PEGRx and PEGRx 2 (Hampton Research, California, USA) as well as Wizard I and II (Emerald Bio). For crystallization, 1 µl protein solution (20 mg ml⁻¹ N-PAS domain, 20 mM Tris–HCl pH 7.5, 200 mM NaCl, 5 mM DTT) was mixed with 1 µl precipitant solution (Table 1 ▸). Different combinations of precipitant, pH and salt were tested and several optimization screens were used, including Detergent Screen, Additive Screen and Silver Bullets (Hampton Research). After optimization, crystals for data collection were obtained at 291 K. The crystals were recovered and immediately flash-cooled in liquid nitrogen.
Data were collected from a single flash-cooled crystal, which was a long rod, using 25%(v/v) glycerol as a cryo­protectant on beamline BL17U at Shanghai Synchrotron Radiation Facility (SSRF; Table 2 ▸). The data were processed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▸ ).