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Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) from Saccharomyces cerevisiae is essential for cell viability. It has been overexpressed in Escherichia coli and has been crystallized at 296 K using polyethylene glycol (PEG) 1500 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 59.48, b = 138.54, c = 157.91 Å, α = β = γ = 90°. Two molecules of trimeric dUTPase from S. cerevisiae are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 3.36 Å3 Da−1 and a solvent content of 63%. The diffraction limit of the crystals could be significantly extended by the crystal-annealing procedure. A set of native data extending to 2.7 Å resolution has been collected at 100 K using synchrotron X-rays.

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