Computational protocol: Cyclic Adenosine 5′-DiphosphateRibose Analogswithout a “Southern” Ribose Inhibit ADP-ribosyl Cyclase–HydrolaseCD38

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Protocol publication

[…] The human CD38 catalytic domain was expressed and purified as described previously., The protein was diluted to 10 mg/mL for crystallization trials. Crystals of wild type CD38 were obtained by the hanging droplet vapor diffusion method with the reservoir buffer in 0.1 M sodium acetate, pH 4.0, 15% PEG 10K, 0.2 M ammonium acetate, and 3% isopropanol. They were harvested and soaked in 0.1 M sodium acetate, pH 4.0, 15% PEG 10K, 16% ethylene glycerol, and 5 mM compound 7 for 1 h at 295 K and then flash-frozen in liquid nitrogen. The diffraction data were collected at 100 K on beamline BL17U at the Shanghai Synchrotron Radiation Facility and processed with HKL2000. Molecular replacement was performed using the program Phaser from the CCP4 suite, and the wild-type human CD38 (PDB code 1YH3) was used as the search model. The model was refined with Refmac and then cycled with manual building in Coot. Hydrolyzed compound 7 was built into positive difference electron-density maps of the CD38 model after a few restrained refinement runs with the stereochemical restraints generated from the program PRODRG. TLS refinement was incorporated into the later stages of the refinement process. Solvents were added automatically in Coot and then manually inspected and modified. The final model was analyzed with MolProbity, showing that 98% residues were in the Ramachandran favored region with only one residue (D202 in chain B) in the Ramachandran outlier region. Data collection and model refinement statistics are summarized in Table 1. The coordinates and structure factors are deposited in the Protein Data Bank with the code 4TMF. […]

Pipeline specifications

Software tools CCP4, Coot, PRODRG, MolProbity
Applications Drug design, Protein structure analysis
Organisms Homo sapiens
Chemicals Adenosine Diphosphate, Ribose