Computational protocol: Structure of the uracil complex of Vaccinia virus uracil DNA glycosylase

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Protocol publication

[…] To prepare a complex of D4 with uracil we used a co-crystallization strategy. Prior to crystallization, we incubated 30 µl D4 (9 mg ml−1 or ∼330 µM in 25 mM HEPES buffer pH 7.3, 0.3 M KCl, 1 mM TCEP) with 3 µl uracil (200 mM stock solution in 100% DMSO) for 1 h at 277 K. The mixture was used for crystallization by the hanging-drop vapor-diffusion method. 1 µl protein solution was mixed with an equal volume of reservoir solution. Rod-shaped and rectangular-shaped crystals with average dimensions of ∼0.18 × 0.12 mm were obtained after 1–2 d at 277 K. The reservoir solution of the crystal used for data collection consisted of 10% PEG 8000, 0.1 M Tris–HCl pH 8.0, 10% DMSO. To increase the DMSO concentration in the crystal for direct cooling, the cover slip containing the hanging drop was sealed over a new reservoir consisting of 10% PEG 8000, 0.1 M Tris buffer pH 8.0, 25% DMSO and the drop was then allowed to equilibrate for 3 d at 277 K. This crystal was flash-cooled directly in liquid nitrogen.Data were collected on a Pilatus 6M detector at the Advanced Photon Source (beamline NE-CAT 24-ID-C) at 100 K. Previously, we noticed possible binding of DMSO near the active site of D4 in crystals that had been soaked in a solution containing DMSO. We therefore collected highly redundant X-ray data at a wavelength of 1.77 Å that should allow the verification of S atoms in an anomalous difference map. An initial data set of 120 oscillation images (1° per image) was collected at a crystal-to-detector distance of 150 mm. To increase the data multiplicity and improve the signal-to-noise ratio for the anomalous signal, two additional partially overlapping sweeps (200 images each, 1° per frame) were added. The three sweeps were then merged and scaled together. The highly redundant data (multiplicity of 17 overall and 10 in the highest resolution shell) were used to maximize the anomalous signal, which allowed the placement of different buffer components (potassium and chloride ions) and several DMSO molecules (five out of a total of ten) based on their anomalous scattering properties (anomalous scattering coefficients Δf′′ at 1.77 Å: K+, 1.39e−; Cl−, 0.91e−; S, 0.74e−). The signal for S atoms in well ordered Cys and Met residues served as an internal control with regard to the quality of the anomalous difference map.Data were processed with XDS (Kabsch, 2010a ,b ) and SCALA (Evans, 2006) in the CCP4 suite (Winn et al., 2011) as part of the RAPD data-collection strategy at NE-CAT (https://rapd.nec.aps.anl.gov/rapd). Data-collection statistics are listed in Table 1. [...] The unit-cell parameters and the diffraction resolution suggested a value of 2.46 Å3 Da−1 for the Matthews coefficient and a solvent content of 50% for 12 subunits of D4 in the asymmetric unit. The crystal structure was solved by molecular replacement with Phaser (McCoy et al., 2007) using the coordinates of one subunit from the D4 structure (PDB entry 4dof) as a search model. The σA-weighted difference electron-density map (mF o − DF c) at the 3σ contour level calculated after initial map fitting and refinement of the protein residues allowed the placement of uracil molecules in the catalytic pockets of all 12 subunits. The positions of the uracil in the active site were verified by the calculation of σA-weighted difference maps (mF o − DF c ≥ 3σ) using REFMAC (Murshudov et al., 2011) omitting the ligands from refinement and map calculation. Initially automatically generated NCS restraints were employed, and in later stages of the refinement we used loose NCS restraints. Prior to the final refinement cycles 1617 water molecules were added to the model at stereochemically appropriate locations in the difference electron-density map (mF o − DF c ≥ 3σ) using Coot (Emsley & Cowtan, 2004). REFMAC (v.5.7) was used for structure refinement and validation was performed using MolProbity (Chen et al., 2010). Refinement statistics are listed in Table 1. The final atomic coordinates and structure factors for the uracil complex have been deposited in the PDB (entry 4lzb). […]

Pipeline specifications

Software tools XDS, CCP4, Coot, MolProbity
Databases RAPD
Applications Small-angle scattering, Protein structure analysis
Organisms Vaccinia virus, Escherichia coli, Homo sapiens
Chemicals Uracil