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).