Computational protocol: Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization

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Protocol publication

[…] The initial crystallization screening was performed with screening kits using the hanging-drop vapour-diffusion method at 288 K. The hanging drops were mixtures of 2 µl reservoir solution and 2 µl protein solution. Crystals of the wild-type protein were grown in 9% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris–HCl pH 8.5 using a protein solution at 30 mg ml−1 in 6–8 weeks. The N253A mutant crystals were obtained in 11% PEG 4000, 0.2 M sodium acetate trihydrate, 0.3 M Tris–HCl pH 8.5, 5% glycerol using a protein solution at 60 mg ml−1 in two weeks. X-ray diffraction data were collected and processed on beamlines BL13B1 and BL15A1 at NSRRC, Hsinchu, Taiwan. The wild-type and N253A crystals belonged to space groups P21 and P212121, respectively. The structures were determined by the molecular-replacement method using the MsTS structure as a template (Caner et al., 2013); the search models were generated by MODELLER (Šali & Blundell, 1993) and the phase problem was solved using Phaser (McCoy et al., 2007). Further refinement was performed with Coot (Emsley et al., 2010) and REFMAC5 (Murshudov et al., 2011), and the Ramachandran analysis was performed using MolProbity (Chen et al., 2010). Noncrystallographic symmetry restraints were applied in structural refinement of the wild type but not in that of the N253A mutant. The statistics of data collection and refinement are summarized in Table 1. Figures were generated using PyMOL (DeLano, 2002). […]

Pipeline specifications

Software tools REFMAC5, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Deinococcus radiodurans
Chemicals Maltose, Sucrose, Trehalose