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Figure 2.
Fig. 2. The structure and topology of MxiC. a, A ribbon
diagram of MxiC, colored from blue at the N terminus to red at
the C terminus. Views rotated by 90° about the long axis are
shown. b, A diagram of the topology of MxiC illustrating the
four-helix X-bundle of each domain colored as for a. c, Two
molecules of MxiC from the P2[1]2[1]2[1] crystal form (molecule
B in magenta and molecule C in cyan), overlaid via their central
domain (residues 154–265), illustrating the extremes of the
movement seen for domains 1 and 3 (shown with cylindrical
helices). Methods: Initial crystallization conditions were
obtained by sparse-matrix screening,^30 using the sitting drop
vapor diffusion technique. Drops were prepared using an OryxNano
crystallization robot (Douglas Instruments) by mixing 0.2 μl of
protein (7 mg ml^− 1 in 20 mM Tris (pH 7.5), 150 mM NaCl) with
0.2 μl of reservoir solution and were equilibrated against 100
μl of reservoir solution at 20 °C. Initial, low-resolution
diffracting crystals of MxiC[FL] grew within two weeks in
condition P2-26 of the PACT Premier screen (0.2 M NaBr, 0.1 M
BisTris–propane (pH 7.5), 20% (w/v) PEG3350: space group
P4[3]2[1]2 with one molecule in the asymmetric unit) and
condition 3 of Molecular Dimensions Structure Screen II (2%
(v/v) dioxane, 0.1 M bicine (pH 9.0), 10% (w/v) PEG20000: two
different, related P2[1] forms with two molecules in the
asymmetric unit). The former condition yielded
diffraction-quality crystals of SeMet-labeled MxiC[FL]†.
Crystals of native MxiC[FL] diffracting to 3.0 Å
resolution grew in 0.2 M Na[2]SO[4], 0.1 M BisTris–propane (pH
6.5), 20% (w/v) PEG3350, again in P4[3]2[1]2 but with a longer c
axis and two molecules in the asymmetric unit. The methylation
reaction was performed as described in Refs. 31 and 32 on
purified MxiC[FL] and MxiC[NΔ73] each at 1 mg ml^− 1 in 50 mM
Hepes (pH 7.5), 250 mM NaCl. Samples were centrifuged (5 min,
13,000 rpm, 10,000g 4 °C) before purification of soluble
methylated protein by size-exclusion chromatography (as
described above). Methylation of all lysine side chains and the
N terminus was verified by mass spectrometry (42,952 Da for
MxiC[FL] and 35,106 Da for MxiC[NΔ73]). The P222 crystal form
grew in 1.0 M succinic acid, 0.1 M Hepes (pH 7.0), 1% (w/v)
PEG2000MME. The P2[1]2[1]2[1] crystal form grew in 0.2 M sodium
acetate, 0.1 M BisTris–propane (pH 7.5), 20% (w/v) PEG3350.
Crystals of MxiC were cryoprotected in reservoir solution
containing 25% (v/v) glycerol for 15 s and flash cryocooled in
liquid nitrogen for data collection. Diffraction data were
recorded at 100 K. Data were indexed and integrated in
MOSFLM,^33 and scaled with Scala,^34 within the CCP4 program
suite,^35 except for the native MxiC[FL] P4[3]2[1]2 3.0 Å
dataset, which was indexed in Labelit^36 and integrated in
XDS,^37 both run from the processing suite Xia2 (G. Winter et
al., unpublished program). Initial phases were computed using
SHARP:^38 five sites were found by SHELXD^39 run from the suite
of programs autoSHARP^40 against F[A]s calculated from the peak,
inflexion and low-energy remote wavelengths of a SeMet-labeled
P4[3]2[1]2 MxiC[FL] crystal. The coordinates and B-factors of
these sites were refined in SHARP against the above data plus
the second remote wavelength from the same SeMet crystal.
Solvent flattening was performed using CCP4-DM^41 and
SOLOMON,^42 yielding a 3.5 Å map that was used for initial
model building guided by the YopN–TyeA structure (PDB ID
1xl3).^5 After alternate cycles of model building in Coot,^43
refinement in Buster-TNT,^44 and simulated annealing in
PHENIX,^45 this initial model was used for molecular
replacement, using CCP4 PHASER,^46 into the higher resolution
P2[1]2[1]2[1] form. The resultant model was used for molecular
replacement against the MxiC[NΔ73] P222 and native MxiC[FL]
P4[3]2[1]2 crystal forms. The final Buster-TNT refinements in
the latter forms used NCS restraints throughout, and extra
geometry restraints tying the geometry to Refmac^47-refined
models, to improve the stereochemistry (as Refmac5 implements
torsion angle restraints and can refine riding H atoms), a
refinement strategy devised by Dr. Stephen Graham (University of
Oxford).
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