Computational protocol: Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations

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Protocol publication

[…] CgGDE crystals were obtained with the sitting-drop method at 20 °C. The reservoir liquid contained 10% poly(ethylene glycol) 5,000 monomethyl ether, 0.1 M Hepes pH 7.0 and 5% tacsimate pH 7.0. Before crystallization, 0.3 mM Tri(2-carboxyethyl)phosphine hydrochloride was added to the protein solution. To produce crystals of the CgGDE-maltopentaose complex, the protein solution was incubated with 100 mM maltopentaose (Sigma-Aldrich) for 1 h on ice before crystallization experiments. For data collection, crystals were equilibrated in the reservoir solution supplemented with 25% ethylene glycol for 30 s, flash-cooled and stored in liquid nitrogen.Diffraction data were collected on an ADSC Q315 charge-coupled device detector at the Shanghai Synchrotron Radiation Facility beamline BL17U, at 100 K (). A MAD data set was collected on a ligand-free SeMet-substituted crystal, at three wavelengths near the Se K-edge (0.9793 Å, 0.9794 Å and 0.9537 Å). Another data set of the ligand-free crystal was collected on a native crystal at 1.0391 Å. The data set for the CgGDE-maltopentaose complex crystal was collected at 0.9792 Å. Diffraction data were scaled with mosflm, integrated with scala and the intensities were converted to structure factors with ctruncate.The ligand-free CgGDE structure was determined by the MAD method using the anomalous signal of Se, with the phenix AutoSol pipeline. After the substructure search step, which found 61 sites (62 expected), the figure of merit was 0.47. Subsequent density modification gave an excellent electron-density map, which allowed building of the entire structure, with coot and O. Using this structure as the search model, the CgGDE-maltopentaose complex structure was determined by the molecular replacement method with molrep. Refinements were carried out with refmac and phenix. An analysis with procheck indicates the ligand-free structure, refined against the native data set, has 84.3% residues in the most-favoured regions in the Ramachandran plot, 15.1% in the additionally allowed regions, 0.6% in the generously allowed regions and no residues in the disallowed regions. The refined CgGDE-maltopentaose complex structure has 84.8, 14.8, 0.3 and 0.1% residues in the most-favoured, additionally allowed, generously allowed and disallowed regions in the Ramachandran plot.Structural homologues of CgGDE were searched with the Dali server. Buried surface area between domains were calculated with areaimol. Mosflm, scala, ctruncate, refmac, procheck and areaimol are programs in the ccp4 suite. […]

Pipeline specifications

Software tools CCP4, PHENIX, Coot, Molrep, PROCHECK, DALI
Application Protein structure analysis
Organisms Homo sapiens
Diseases Metabolism, Inborn Errors, Machado-Joseph Disease
Chemicals Glucose