Computational protocol: Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR

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Protocol publication

[…] Crystals were obtained by hanging-drop vapor diffusion by mixing equal volumes of protein (5–30 mg/ml) and reservoir solution (0.1 M Tris-Cl [pH 8.0], 2.9 M NaCl, 15% xylitol) followed by incubation at 20 °C. Crystals appeared within 1 to 2 d, with typical dimensions of 0.2 mm × 0.2 mm × 0.2 mm. Crystals were flash-frozen in liquid nitrogen and kept at 100 K during data collection.Crystallographic statistics for data collection are shown in . Datasets were collected using synchrotron radiation at the Cornell High Energy Synchrotron Source (CHESS, Ithaca, beamline A1). Data reduction was carried out with the software package HKL2000 []. The space group was determined to be C2 with a = 144.5 Å, b = 72.8 Å, c = 106.1.4 Å, and β = 110.8°. The asymmetric unit consists of two molecules, bound to four molecules of c-di-GMP and two magnesium ions. Phases were obtained from molecular replacement with the isolated GGDEF and CheY domains of PleD (PDB code: 1W25) [] as search models using the software package Phenix []. The model of the WspR dimer was built manually starting from the molecular replacement solution. Refinement using CNS [] and O [] yielded the final model. Illustrations were made in Pymol (DeLano Scientific). […]

Pipeline specifications

Software tools PHENIX, CNS, PyMOL
Application Protein structure analysis