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Figure 1.
Figure 1. The overall tertiary structure of the
CST-benzamidine complex. CST was co-crystallized with
benzamidine by equilibrating droplets containing 0.62 mM CST, 30
mM MgSO[4], 0.6 mM CaCl[2], 6.0 mM benzamidine, 50 mM Tris-HCl
buffer (pH 8.15) against reservoir solutions containing 1.4 M
MgSO[4] and 0.1 M Tris-HCl buffer (pH 8.5). A crystal adopting
the form of a rectangular prism (0.15 mm×0.15 mm×0.3
mm) was obtained after four days. X-ray data were collected at
room temperature on a DIP 2030H image plate detector (Mac
Science Co., Ltd), using double-mirror focusing optics and Cu Ka
radiation (l=1.5418 Å) generated by a Rigaku rotating
anode generator RU-200BH operating at 45 kV and 75 mA. The data
were processed and reduced with the programs Denzo &
Scalepack.[22.] The structure of CST was determined by molecular
replacement using AMoRe. [23.] Strong solutions were obtained
from a rotation function (cc=0.38) and translation function
(cc=0.65) calculated over the resolution range 20-4.0 Å
using AST as the search model (PDB accession number 2TBS).
Following rigid-body refinement (R-factor (20-3.0
Å)=0.28), the model was rebuilt using the program
XFIT.[24.] Seven sequence differences with AST at positions 28,
125, 152, 153, 170, 235, and 244 and the deletion of two
C-terminal residues were incorporated into the model of CST.
Conventional crystallographic refinement was carried out using
CNS. [25.] Water molecules were added to the model where the
difference density exceeded 4s, and the peaks were within a
reasonable distance of hydrogen-bonding partners on the enzyme
(<3.3 Å). The statistics of the diffraction data and the
final refinement cycle are listed in Table 1. Figure 1 and
Figure 2 were prepared using MOLSCRIPT [26.] and Raster3D. [27.]
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