X-ray structural study of modB.

The modB-containing fraction from HiTrap SP was further purified by gel filtration. A Superdex 200 (10/300) column (GE Healthcare) was equilibrated with PBS, then the single peak at 12.6 ml (~135 kDa) was pooled and concentrated with an Amicon ultra device (30,000 MWCO; Merck Millipore) to 7.2 mg/ml. Rod-shaped clustered crystals were grown from the Index #90 (0.2 M sodium formate, 20% [w/v] PEG 3350) (Hampton Research) condition, and we obtained single crystals by microseeding. The crystals were transferred to cryoprotectant solution (20% [v/v] glycerol, 0.16 M sodium formate, 16% [w/v] polyethylene glycol [PEG] 3350) for 10 seconds and then flash-cooled by liquid nitrogen and stored. The x-ray diffraction data of the modB crystals was collected at beamline AR-NE3a in Photon Factory using a wavelength of 1,000 Å by an ADSC Q270 CCD detector. The crystal was kept at 100 K during the data collection. The data were processed with iMosflm (58), and the molecular replacement solution was calculated by the Molrep software program (58) using the human HexB structure (20) as a search model. The structural refinement was performed by the Refmac5 software program (58), and the model was manually fixed to modB sequences using the Coot program (59). The Ramachandran plot analysis of modB by the Rampage software program (58) showed 465 residues (95.7%) in favored regions, 21 residues (4.3%) in allowed regions, and none in outlier regions. The data collection and the structural refinement statistics are summarized (Supplemental Table 2).