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Figure 1.
Figure 1. The three-dimensional structure of Mtb-IPMDH. (a)
C^a representation of the four subunits of Mtb-IPMDH found in
the asymmetric unit. Each subunit is represented by a different
colour (A, red; B, green; C, yellow; D, cyan). The six local
dyads and the approximate local dyads are indicated. The
resemblance of the arrangement to a D[2] tetramer is clearly
discernible. (b) Ribbon representation of the structure of the
homodimer of Mtb-IPMDH. The two domains in one subunit are
identified by different colours, domain 1 is displayed in red,
domain 2 in blue. The two different shades of red in domain 1
denote the two sub-domains (see the text). (c) Ribbon
representation of the structure of one subunit of Mtb-IPMDH. The
secondary structure elements are coloured with a-helices in red
and b-strands in yellow. The N and C termini are labelled. The
1.0 kb gene fragment of Rv2995c was amplified by PCR from M.
tuberculosis H37Rv genomic DNA with the following
oligonucleotides (obtained from MWG Biotech):
5'-AAAATCATGAGCAAACTCGCGATCATTGCCGGTGACGGGATCGGGC-3' and
5'-AAAACTCGAGTTAGAGCGCGGCGGCAATCCGTTCGCCGACA-3' as forward and
reverse primers, respectively. In the forward primer, the
additional underlined bases were introduced to get the gene
in-frame for transcription. Therefore, the gene product obtained
contains an extra serine residue at position 2 (codon AGC). The
amplified fragment containing 5'-BspHI and 3'-XhoI restriction
sites (shown in bold in the primer sequence) was cloned into
PCR-Blunt TOPO vector (Invitrogen). The construct was digested
with BspHI and XhoI, and the insert fragment was subcloned into
the pETM-11 expression vector digested with the restriction
enzymes NcoI and XhoI. Both TOPO and the final pETM-11
constructs were sequenced to confirm the cloning of the leuB
gene sequence in-frame. Expression of the 35 kDa protein was
carried out in E. coli BL21 Star (DE3) pRARE cells. These cells
were prepared by transforming BL21 Star (DE3) cells (Invitrogen)
with the pRARE plasmid isolated from the Rosetta strain
(Novagen). Cells from an overnight pre-culture were grown in LB
broth medium containing kanamycin (30 µg/ml) and
chloramphenicol (10 µg/ml) at 310 K and 200 rpm. The
culture was induced with 0.2 mM isopropyl
b-D-thiogalactopyranoside (IPTG) at an A[600] of approximately
0.6 at 293 K. Following induction, the culture was incubated for
about 15 hours at 293 K and 200 rpm and then harvested. The
cells were stored at 193 K until further processing. One gram of
cell pellet was lysed by sonication in 30 ml of buffer A (50 mM
Tris (pH 8.0), 200 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol,
2 mM b-mercaptoethanol (b-ME)) for 20 minutes in 25 second
pulses at 4 °C. The cell debris was pelleted by
centrifugation for 30 minutes at 4 °C and 20,000 rpm. The
crude lysate was filtered through a 0.2 µm pore size
membrane and loaded onto a 5 ml Hi-Trap Chelating HP column
(Amersham Biosciences) charged and equilibrated with Ni2+ and
buffer A, respectively. In order to remove unbound proteins, the
column was first washed with 25 ml of buffer A, then with 25 ml
of buffer B (50 mM Tris (pH 8.0), 600 mM NaCl, 10 mM imidazole,
5% (v/v) glycerol, 2 mM b-ME) and then with 25 ml of buffer C
(50 mM Tris (pH 8.0), 200 mM NaCl, 50 mM imidazole, 5% (v/v)
glycerol, 2 mM b-ME). The protein was eluted by running a linear
gradient from 50 mM to 800 mM imidazole (in buffer C) in a total
volume of 50 ml. The peak fractions were pooled and dialysed
against buffer D (50 mM Tris (pH 8.0), 200 mM NaCl, 5% (v/v)
glycerol, 2 mM b-ME). During dialysis overnight at 277 K, the
His tag was cleaved off by adding recombinant tobacco etch virus
(TEV) protease. The cleaved and dialysed protein mixture was
passed through a Ni-NTA column pre-equilibrated with buffer D.
The protein in the flow-through was subsequently purified by
gel-filtration (Superdex 200 16/60, Amersham Biosciences) using
buffer D for both equilibration and elution. The protein eluted
with an apparent molecular mass of approximately 70 kDa, which
indicated the presence of a homodimer in solution. The peak
fractions analysed by SDS-PAGE were pooled, and concentrated to
10 mg/ml. The purity of the protein was estimated by SDS-PAGE
and by dynamic light-scattering. The initial crystallization
screening was done in Greiner 96-well plates using the
sitting-drop, vapour-diffusion method (1 µl of protein and
1 µl of Hampton crystal screens 1 and 2). A few needles
were observed in condition #32 of screen #2 (1.6 M
(NH[4])[2]SO[4], 0.1 M Hepes (pH 7.5), 0.1 M NaCl) at 293 K.
This condition was optimized to 2.0 M (NH[4])[2]SO[4], 0.1 M
Hepes (pH 8.0), 0.1 M NaCl. The crystallization experiments
using the optimized condition as reservoir solution were
performed in 24-well plates using the hanging-drop method with
1.5 µl of protein and 1 µl of reservoir solution at
293 K. Crystals were typically obtained within 48 hours and grew
up to 1.0 mm in size. The crystals belong to space group
P2[1]2[1]2[1] with unit cell dimensions of a=78.47 Å,
b=97.06 Å and c=181.91 Å, and have four molecules in
the asymmetric unit. They diffract X-rays to about 1.5 Å
resolution. Crystals were treated with cryoprotectant (22% (v/v)
glycerol in reservoir solution) and diffraction data were
collected at 100 K on the EMBL wiggler beamline BW7B (DESY,
Hamburg, Germany) using a MAR345 mm imaging plate detector. Two
sweeps of data were collected: a low-resolution sweep (180°)
extending to 3.0 Å resolution and a high-resolution sweep
(180°) extending to 1.65 Å resolution. The data were
indexed and integrated using DENZO23 and scaled using
SCALEPACK.23 The redundancy-independent merging R-factor
R[r.i.m.] as well as the precision-indicating merging R-factor
R[p.i.m.]24 were calculated using the program RMERGE (available
from http://www.embl-hamburg.de/ msweiss/projects/msw_qual.html
or from M.S.W. upon request). The relevant data processing
parameters are given in Table 2. Intensities were converted to
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