2.1. Protein preparation and crystallization
The gene for CMY-10 was chemically synthesized and subsequent subcloning and purification were performed as described previously (Lee et al.
). Crystals of CMY-10 were grown in a precipitant solution consisting of 18% polyethylene glycol 8000, 0.1 M
sodium cacodylate pH 6.5, 0.2 M
zinc acetate dehydrate using the microbatch crystallization method.
gene of Thermococcus onnurineus
NA1 was cloned into pET22b-CPD 10H, an in-house-modified form of pET22b (Novagen), to express a protein fused to His10
-tagged CPD (cysteinyl protease domain) at the C-terminus (Shen et al.
). The fusion protein was expressed in Escherichia coli
BL21 (DE3) RIPL strain (Novagen) at 310 K. Bacterial lysates were prepared by sonication in buffer A
composed of 20 mM
Tris–HCl pH 7.5, 100 mM
NaCl, 5 mM
β-mercaptoethanol. Cleared lysates isolated by centrifugation were loaded onto a column packed with HisPur Cobalt Resin (Thermo) and washed with buffer A
containing 10 mM
imidazole. On-gel auto-cleavage of His10
-tagged CPD was performed by incubating the resin with buffer A
containing 100 mM
phytate for 2 h at room temperature, which activates the protease activity of CPD. TON_0340 was eluted with buffer A
and further purified with a HiTrap Q HP column (GE Healthcare). For crystallization, the final sample was concentrated to 9 mg ml−1
in a buffer solution composed of 20 mM
Tris–HCl pH 7.5, 100 mM
NaCl, 1 mM
dithiothreitol. Crystals were obtained by the hanging-drop vapour-diffusion method using a precipitant solution consisting of 5%(v
) 2-propanol, 100 mM
sodium acetate pH 5.0, 350 mM
zinc acetate at 295 K.
Hen egg-white lysozyme was purchased from BIO Basic Inc. and was used without further purification. Lysozyme crystals were grown at 295 K using the microbatch crystallization method. Small drops composed of 1 µl protein solution (20–50 mg ml−1) and an equal volume of a precipitant solution consisting of 1 M NaCl, 0.1 M citric acid pH 4.0 were pipetted under a layer of a 1:1 mixture of silicon oil and paraffin oil in 72-well HLA plates (Nunc).
2.2. Data collection
A 2.1 Å resolution MAD data set for CMY-10 was collected at wavelengths of 1.2825 Å (peak), 1.2828 Å (inflection point) and 1.1700 Å (high-energy remote) using an ADSC Quantum 270 CCD on the microfocus beamline PF-17A at the Photon Factory, Japan. For each data set, 180 diffraction images with a 1° oscillation width were collected with the crystal-to-detector distance set to 180 mm (Table 1).
Data-collection, refinement and phasing statistics
A 2.3 Å resolution SAD data set for TON_0340 was collected at the zinc absorption peak using an ADSC Quantum 315 CCD on beamline 4A of Pohang Light Source, Republic of Korea (Table 1). In order to avoid spot overlaps arising from the long c axis of the unit cell, we used a 90°-bent metal pin to align the c axis along the spindle axis and collected 200 images of 1° oscillation with the crystal-to-detector distance set to 300 mm (covering 200° of oscillation) that are free of interference from the metal pin (Table 1).
A 1.8 Å resolution SAD data set for hen egg-white lysozyme was collected at a wavelength of 1.2829 Å using an ADSC Quantum 270 CCD on the microfocus beamline PF-17A at the Photon Factory, Japan. A total of 180 frames of 1° oscillation were collected with the crystal-to-detector distance set to 176 mm (Table 1).
2.3. Data processing and phasing
Diffraction data were processed and scaled using DENZO
from the HKL
-2000 program suite (Otwinowski & Minor, 1997
). Experimental phasing of CMY-10 and lysozyme was performed with the AutoSol
program (Terwilliger et al.
) in the PHENIX
suite (Adams et al.
), which is an experimental phasing pipeline that combines HySS
(Hybrid Substructure Search
; Grosse-Kunstleve & Adams, 2003
) for finding heavy-atom sites, Phaser
(McCoy et al.
) or SOLVE
) for calculating experimental phases and RESOLVE
) for density modification and model building. Experimental phasing of TON_0340 was performed with autoSHARP
(Vonrhein et al.
), an automatic structure-solution system that includes the heavy-atom refinement and phasing program SHARP
(de La Fortelle & Bricogne, 1997
), the density-modification program SOLOMON
(Abrahams & Leslie, 1996
) and the ARP
package (Perrakis et al.
) for automated model building and refinement. The auto-built models from the phasing programs were completed using Coot
(Emsley & Cowtan, 2004
) and refinement was performed with a maximum-likelihood algorithm implemented in CNS
(Brünger et al.