Computational protocol: Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

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Protocol publication

[…] The cUNG–Ugi complex was crystallized using the hanging-drop method at 4°C with 7.5 mg ml−1 protein complex in 25 mM Tris–HCl pH 7.5, 10 mM NaCl, 1 mM EDTA. Drops were made by mixing 1 µl protein with 0.2 µl 0.1 M NaBr and 0.8 µl reservoir solution consisting of 0.1 M Tris–HCl pH 7.4, 270 mM Li2SO4, 4% PEG 550 MME, 17% PEG 4000. A crystal of about 200 × 200 × 50 µm in size was transferred to a cryoprotectant solution made up of 17% PEG 4000, 10% glycerol and the other reservoir additives at their original concentrations and was then flash-cooled in liquid nitrogen.Diffraction data were collected on beamline ID-29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France (de Sanctis et al. 2012) using an ADSC Quantum 315r detector. The crystal belonged to space group P21, with unit-cell parameters a = 98.21, b = 86.92, c = 175.37 Å, β = 90.35°, with pseudosymmetric translation (x + 0.17, y + 1/2, z + 0.17) and twinning (−h, −k, l; twin fraction 0.235). There are eight complexes (16 molecules) in the asymmetric unit, giving a solvent content of 54.8% and a Matthews coefficient of 2.7 Å3 Da−1.The data were indexed, integrated, scaled and converted to structure factors using the XDS program package (Kabsch, 1993). Two related but not congruent lattices were observed in the data, resulting in many overlapped reflections. It was necessary to decrease the value of the WFAC1 parameter in XDS in order to increase the number of misfits rejected before scaling and merging of the data. The structure was solved by molecular replacement using MOLREP (Vagin & Teplyakov, 2010) in CCP4 without the PST vector information. The search model was a model of the cUNG–Ugi complex made by superimposing cUNG (PDB entry 1okb; Leiros et al., 2003) on the structure of the hUNG–Ugi complex (PDB entry 1ugh; Mol, Arvai, Sanderson et al., 1995). The structure was refined in REFMAC5 (Murshudov et al., 2011) using amplitude-based twin refinement (and no TLS refinement) interspersed with rounds of manual model building in Coot (Emsley et al., 2010). Automatically generated NCS restraints were used in the first ten refinement cycles only, whereas twin refinement was used in all steps. The final model had R work and R free values of 23.7 and 28.3%, respectively, with acceptable geometry, and was validated using MolProbity (Chen et al., 2010). Details of the data-collection and refinement statistics are given in Table 1.The UNG–Ugi interfaces of cUNG–Ugi, hUNG–Ugi (PDB entry 1ugh) and herpes simplex virus 1 UNG–Ugi (PDB entry 1udi; Savva & Pearl, 1995) were analyzed with the Protein Interfaces, Surfaces and Assemblies service (PISA; Krissinel & Henrick, 2007). In addition, the WHAT IF web interface (Vriend, 1990) was used to identify interface electrostatic interactions with interatomic distances of <6 Å. The figures were generated using PyMOL ( and the electrostatic surface potential was calculated using the APBS plugin in PyMOL (Baker et al., 2001). The accessible surface area (ASA) was calculated with Surface Racer 5.0 (Tsodikov et al., 2002) with a probe radius of 1.40 Å. cUNG (chain E) and hUNG (chain E) from the UNG–Ugi complexes were used. […]

Pipeline specifications

Software tools MISFITS, Molrep, REFMAC5, MolProbity, PyMOL
Applications Phylogenetics, Protein structure analysis
Organisms Gadus morhua, Homo sapiens
Chemicals Uracil