MATERIALS AND METHODS
Purification of recombinantly expressed GmCHS1
Heterologous expression and purification of GmCHS1 were reported previously.2 Briefly, the GmCHS1 ORF was introduced into pCold I vector to express the GmCHS1 with anN -terminal extension including His6-tag. TheN -terminal extension sequence includes MNHKVHHHHHHIEGRHM , where the underlined Met indicates the original initiating Met of GmCHS1. The resultant plasmid was introduced into Escherichia coli BL21 strain. The transformed E. coli was cultured in LB medium containing 100 µg/mL ampicillin and the expression of GmCHS1 was induced by cold-shock induction. The harvested cells were disrupted and the lysate was centrifuged at 100,000 × g for 1hr at 4˚C. The supernatant from the centrifugation was applied to a column filled with Ni-NTA agarose (5 mL; Qiagen, Hilden, Germany) equilibrated with 50 mM Tris-HCl (pH 7.5) (buffer A) containing 150 mM NaCl. Then, proteins were eluted with buffer A containing 200 mM imidazole. The eluted GmCHS1 was then applied to a size-exclusion column (Superdex 200 increase 10/300, GE Healthcare, Chicago, IL, USA).
Crystallization, data collection and structure determination
GmCHS1 crystals were grown at 20°C by vapor diffusion, mixing 1 μL protein solution (6.7 mg/mL) with 1 μL well solution containing 30% PEG4000, 0.2M ammonium acetate and 0.1M sodium citrate (pH 5.6). The typical crystals grew to needle-like structures with 0.5 mm length in average within 1 week. GmCHS1-naringenin complex was prepared by incubating an apo-GmCHS1 crystal with naringenin (Nacalai tesque, Kyoto, Japan) with 2 mM final concentration for 1 hr at 20°C prior to be flash-frozen in liquid N2. X-ray diffraction data were collected at wavelengths of 1.00000 Å using an EIGER 9M detector (DECTRIS Ltd., Baden-Daettwil, Switzerland) on BL32XU beamline installed ZOO system, SPring-8, Harima, Japan.5 These completed reflection data were collected using a helical data collection scheme, and were indexed and integrated with the KAMO pipeline, using DIALS.6,7 Integration intensities and merging were performed using XSCALE with outlier rejections implemented in KAMO.8 Structures were solved by molecular replacement using MOLREP with Pinus sylvestris CHS (PDB ID: 6DXA) as a search model.9 GmCHS1 crystallized with two molecules in the asymmetric unit. Manual model building and refinement of both structures were performed using Coot and REFMAC.10,11 3D molecular images were created using the PyMOL program.12 Analysis of interior cavities and surface pockets were performed by the CASTp 3.0 server program and the result was visualized by the PyMOL program.13 The atomic coordinates and structural factors have been deposited in the Protein Data Bank with PDB codes 7BUS (apo) and 7BUR (complex).