Computational protocol: O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

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Protocol publication

[…] See for expression and purification details of human OGT (312-1031). Protein was crystallized in complex with donor substrate/product, and crystals were soaked with peptide/glycopeptide prior to freezing. Vapor diffusion crystallization experiments with hanging drops containing 1 μl protein (100 μm in a buffer of 10 mm Tris-Cl pH 8.5, 50 mm NaCl, 0.5 mm THP, 1 mm UDP or UDP-5S-GlcNAc) and 0.6 μl reservoir solution (1.45 m K2HPO4, 10 mm EDTA, 1 % xylitol) gave bar-shaped crystals with maximum dimensions of 0.1 × 0.1 × 0.4 mm after 3—4 days at 20 °C. These were transferred to a drop of reservoir solution containing 2 mm of the (glyco)peptide (Ac-PVSVPYS(-β-O-GlcNAc)SAQSTS-NH2) for 30 min, then cryoprotected (1.45 m K2HPO4, 10 mm EDTA, 27 % xylitol) and flash-frozen. Data were collected at the European Synchrotron Radiation Facility (ESRF) at 100 K and wavelengths of 0.939 Å and 0.873 Å on beamlines ID14-4 and ID23-2, respectively. Crystals belonged to space group P321 and contained 4 molecules per asymmetric unit. The structure was solved by molecular replacement using the A chain of PDB ID 3PE3 as the search model. Model building was performed in Coot, and various programs of the CCP4 suite, were used for structure refinement. Ligand topologies were calculated using PRODRG. Data collection and refinement statistics are given in . […]

Pipeline specifications

Software tools Coot, CCP4, PRODRG
Applications Drug design, Protein structure analysis
Organisms Homo sapiens
Chemicals Nucleotides, Phosphates, Glycosylphosphatidylinositols