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PDBsum entry 3c0v
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Plant protein
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PDB id
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3c0v
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References listed in PDB file
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Key reference
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Title
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Mad phasing using the (ta6br12)2+ cluster: a retrospective study.
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Authors
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O.Pasternak,
A.Bujacz,
J.Biesiadka,
G.Bujacz,
M.Sikorski,
M.Jaskolski.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2008,
64,
595-606.
[DOI no: ]
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PubMed id
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Abstract
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The crystal structure of cytokinin-specific binding protein (CSBP) containing
four independent molecules with 4 x 155 = 620 residues in the asymmetric unit of
the P6(4) unit cell has been solved by three-wavelength MAD using 1.8 angstroms
resolution data recorded from a crystal derivatized with the
dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a
very strong anomalous signal (allowing successful phasing even using peak SAD
data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the
asymmetric unit have low occupancy (about 0.3). The derivative structure has
been successfully refined to R = 0.158, providing interesting details on the
geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on
the backsoaking of a cytokinin ligand that was originally part of a
CSBP-cytokinin complex in the native crystals used for (Ta6Br12)2+
derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+
ions at artificially imposed resolution limits shows that it is not possible to
resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9
angstroms. Additionally, for successful Ta identification the (Ta6Br12)2+
complex should be specifically bound and ordered. Good binding at the protein
surface is facilitated by the presence of acidic groups, indicating higher pH
buffer conditions to be preferable. In addition, the water channels in the
crystal should be sufficiently wide (at least 11 angstroms) to allow free
diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the
initial molecular-replacement calculations provides interesting insights into
how the peculiar packing mode and strong bias of the
molecular-replacement-phased electron-density maps had hindered successful
solution of the structure by this method.
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Figure 4.
Figure 4 Anomalous difference map calculated for the best
Ta[6]Br[12] cluster, TBR1, using phases generated by SOLVE and
subsequent density modification for Ta-MAD data truncated at
different d[min] levels: 1.8 Å (a), 2.6 Å (b) and
2.9 Å (c). The maps were contoured at levels of 18  for
(a) and (c) or 6 for
(b). (b) must be contoured at a lower level
to emphasize its features or it would appear to be spherical. In
(c), lowering of the contour level does not reveal any real
features. The dark balls indicate the Ta positions located
automatically by SOLVE.
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Figure 6.
Figure 6 (Ta[6]Br[12])^2+-binding sites. The four CSBP
molecules arranged as in the crystallographic asymmetric unit
are coloured green (A), blue (B), red (C) and yellow (D). The
(Ta[6]Br[12])^2+ clusters are marked using orange Ta spheres and
violet Br spheres. (a) An overview of the relation between the
four CSBP molecules and the five (Ta[6]Br[12])^2+ ions. The
symmetry-related protein molecules that complete the
(Ta[6]Br[12])^2+-binding sites have been omitted for clarity.
(b) Close-up view of the binding details of two representative
(Ta[6]Br[12])^2+ ions. The binding site of the TBR2 ion is shown
on the left. The same interactions are observed for the TBR1
binding site. The binding site of the TBR4 ion is shown on the
right. A similar environment is observed for TBR5. The
amino-acid residues located in the vicinity of the
(Ta[6]Br[12])^2+ ions are shown in stick representation.
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2008,
64,
595-606)
copyright 2008.
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Secondary reference #1
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Title
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Crystal structure of vigna radiata cytokinin-Specific binding protein in complex with zeatin.
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Authors
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O.Pasternak,
G.D.Bujacz,
Y.Fujimoto,
Y.Hashimoto,
F.Jelen,
J.Otlewski,
M.M.Sikorski,
M.Jaskolski.
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Ref.
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Plant Cell, 2006,
18,
2622-2634.
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PubMed id
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