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Figure 1.
Figure 1. Stereo Figure of the structure of Va85.33. (a)
2F[o] - F[c] omit map in the region around CDR1. (b)
Superposition of the C^a backbone traces of the two Va85.33
dimers in the asymmetric unit. The backbone is colored according
to atomic displacement parameters (blue = 18 Å2, RED = 100
Å2). Superpositions were done with the program
SPDBViewer.[50] The complementarity determining regions, the
fourth hypervariable region (HV4) as well as the C and N termini
are indicated. All Figures were generated with Bobscript, [51]
gl_render (L. Esser, University of Texas Southwestern Medical
Center, unpublished) and PovRay (Persistence of Vision Ray
Tracer, v3.02, POV-Team, www.povray.org). Methods: the Va85.33
domain containing a His[6]-tag was expressed as a secreted
protein in Escherichia coli as described.[35] Crystals were
obtained at 20 °C by vapor diffusion from drops containing 3
µl of protein (5 mg ml -1 in 50 mM Tris-HCl (pH 8.0), 100
mM NaCl) plus 3 µl of reservoir solution (100 mM sodium
citrate-HCl, 1.4-1.7 M lithium chloride, pH 5.0-6.0)
equilibrated against 1 ml of reservoir solution. Hexagonal
crystals appeared after three to ten days and grew to a final
size of 0.7 mm diameter and 0.3 mm thickness within one to three
weeks. Va85.33 crystallized with the symmetry of space group
P3[2]21 with cell constants of a = b = 83.5 Å, c = 132.1
Å, and four molecules per asymmetric unit. Prior to data
collection, the crystals were cryo-protected by transferring
them into harvesting solution (100 mM sodium citrate-HCl, 2 M
lithium chloride, pH 5.5) supplemented with up to 40 % (v/v)
glycerol and flash-cooled in liquid propane. The crystals
diffracted to 1.85 Å Bragg spacing when using synchrotron
radiation. The structure was solved by multiple anomalous
dispersion (MAD) using a seleno-methionine variant (two
methionine residues per molecule). The seleno-methionine variant
of Va85.33 was expressed in the methionine-auxotroph E. coli
strain B834 grown in minimal medium supplemented with the
natural amino acids and seleno-methionine. Purification and
crystallization behavior was essentially unchanged compared to
native Va85.33. The MAD experiment was carried out at beamline
19-ID (SBC-CAT) at the Advanced Photon Source (Argonne National
Laboratory, Argonne, Illinois, USA). Data were indexed,
integrated and scaled with the HKL2000 program package.[52]
Eight selenium sites were identified at 2.5 Å resolution
by direct methods (Shake'n'Bake 2.0[53]) using the data set
collected at the energy for the selenium absorption peak.
Selenium parameters were refined and the resulting phases
(figure of merit 0.49) were improved by density modification
using programs from the CCP4 package, [54] resulting in a figure
of merit of 0.69. Model building was done with the program O.
[55] Structure refinement was carried out with the program CNS
v0.5 [56] employing cycles of simulated annealing, conjugate
gradient minimization and calculation of individual atomic
displacement parameters. Calculation of overall anisotropic
displacement parameters and bulk solvent correction was used
throughout. No non-crystallographic symmetry restraints were
used at the highest resolution (1.85 Å). Water molecules
were added where stereochemically reasonable after the protein
part was completed. Side-chains with poorly defined density were
truncated to alanine for refinement purposes. The final model
contains residues 2 to 112 for molecule 1, residues 2 to 110 for
molecule 2, residues 2 to 112 for molecule 3, residues 2 to 110
for molecule 4, three chloride ions, three glycerol molecules
and 266 water molecules. The correctness of the model was
confirmed through simulated annealing omit maps. The R[free]
value is 23.6 % and the R[work] value is 21.9 % (Table 1).
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