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Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana.

Bajaj M, Moriyama H - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

Bottom Line: Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant.The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 69.90, b = 70.86 A, c = 75.55 A.Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V(M) of 1.8 A3 Da(-1).

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Affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304, USA.

ABSTRACT
The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 A resolution using Cu K alpha radiation. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 69.90, b = 70.86 A, c = 75.55 A. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V(M) of 1.8 A3 Da(-1).

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A crystal of dUTPase from A. thaliana.
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fig1: A crystal of dUTPase from A. thaliana.

Mentions: The initial crystallization conditions were obtained using a screening kit from Hampton Research (Aliso Viejo, CA, USA) by the hanging-drop vapour-diffusion method. Using the EasyXtal Tool (Qiagen, Valencia, CA, USA), 1 µl screening solution was mixed with 1 µl protein solution (10 mg ml−1 protein and 50 mM Tris–HCl pH 7.4) and equilibrated against 1 ml of the same screening solution at 298 K. Two weeks after the initial screening, we found 23 clear drops, 22 drops with heavy precipitation, one drop with phase separation and three drops with an indeterminate number of oil drops. Only one condition out of 50 yielded crystals. These were small plate-like colorless protein crystals stacked upon one another. The reservoir was composed of 2 M ammonium sulfate and 0.1 M Tris–HCl pH 8.5. Using additional screening kits (Hampton Research) and varying the pH, the primary crystallization conditions were refined. Rod-shaped crystals appeared after mixing 1 µl primary screening solution at pH 9.0 with 0.5 µl 0.1 M taurine and 1 µl protein solution. The rod-shaped crystals appeared after two weeks and grew to approximate dimensions of 0.4 × 0.1 × 0.1 mm within one month (Fig. 1 ▶).


Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana.

Bajaj M, Moriyama H - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

A crystal of dUTPase from A. thaliana.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335013&req=5

fig1: A crystal of dUTPase from A. thaliana.
Mentions: The initial crystallization conditions were obtained using a screening kit from Hampton Research (Aliso Viejo, CA, USA) by the hanging-drop vapour-diffusion method. Using the EasyXtal Tool (Qiagen, Valencia, CA, USA), 1 µl screening solution was mixed with 1 µl protein solution (10 mg ml−1 protein and 50 mM Tris–HCl pH 7.4) and equilibrated against 1 ml of the same screening solution at 298 K. Two weeks after the initial screening, we found 23 clear drops, 22 drops with heavy precipitation, one drop with phase separation and three drops with an indeterminate number of oil drops. Only one condition out of 50 yielded crystals. These were small plate-like colorless protein crystals stacked upon one another. The reservoir was composed of 2 M ammonium sulfate and 0.1 M Tris–HCl pH 8.5. Using additional screening kits (Hampton Research) and varying the pH, the primary crystallization conditions were refined. Rod-shaped crystals appeared after mixing 1 µl primary screening solution at pH 9.0 with 0.5 µl 0.1 M taurine and 1 µl protein solution. The rod-shaped crystals appeared after two weeks and grew to approximate dimensions of 0.4 × 0.1 × 0.1 mm within one month (Fig. 1 ▶).

Bottom Line: Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant.The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 69.90, b = 70.86 A, c = 75.55 A.Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V(M) of 1.8 A3 Da(-1).

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304, USA.

ABSTRACT
The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 A resolution using Cu K alpha radiation. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 69.90, b = 70.86 A, c = 75.55 A. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V(M) of 1.8 A3 Da(-1).

Show MeSH