Crystallization experiments were conducted in 48-well plates by the hanging-drop vapor diffusion method at 293 K, and each hanging drop was prepared by mixing 1.0 μl each of protein solution and reservoir solution. Initial crystallization trials yielded some small crystals from the polyethylene glycol (PEG)/Ion crystallization screen (Hampton Research). Crystal quality was improved by the microseed matrix screening method (41), and the seed stock was made from the initial crystals. Ultimately, diffraction-quality crystals were grown in hanging drops at 21°C by mixing 1 μl of protein [16 mg/ml in 25 mM tris-HCl (pH 7.5) and 100 mM NaCl] with an equal volume of 0.1 M MES monohydrate (pH 6.0), 0.05 M CaCl2, and 45% PEG-200. Crystals belonged to the space group P 3 2 1, with unit cell dimensions a = 121.4 Å, b = 121.4 Å, c = 32.4 Å, β = 90°. Complex crystals were obtained by cocrystallization with GDP-Fucose. The GDP-Fucose was dissolved to a final concentration of 2 mM in the protein solution for 2 hours at 4°C before setting up hanging drop crystallization experiments as described above.

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