For x-ray diffraction experiments, crystals were fished out from the crystallization drop using a nylon loop, soaked briefly in a cryoprotectant solution of the crystallization solution supplemented with 30% (v/v) ethylene glycol and flash-cooled in liquid nitrogen. X-ray diffraction datasets were collected on beamline BL17U and BL19U at the Shanghai Synchrotron Research Facility. All diffraction data were indexed, integrated, and scaled using HKL-2000.

Initial phases for each structure were determined by molecular replacement. The structure of apo-M32 was solved using the program BALBES with the Auto-Rickshaw pipeline. The structure was completed with alternating rounds of manual model building with Coot and refinement with REFMAC5 in CCP4suite. The structure of the M32-substrate complex was determined by molecular replacement with the program MOLREP using the apo-M32 as a search model. The structure of GDP-Fucose was built with Coot Ligand Builder, and restraints were created using PRODRG. Iterative model building was performed with Coot software, and refinement was carried out with REFMAC5 in CCP4suit. The final models were evaluated by PROCHECK (42). Data collection and refinement statistics are provided in table S5. PyMOL (DeLano Scientific; was used to produce molecular graphics renditions.

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