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PDBsum entry 2j4d
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DNA binding protein
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PDB id
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2j4d
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References listed in PDB file
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Key reference
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Title
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Cryptochrome 3 from arabidopsis thaliana: structural and functional analysis of its complex with a folate light antenna.
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Authors
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T.Klar,
R.Pokorny,
J.Moldt,
A.Batschauer,
L.O.Essen.
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Ref.
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J Mol Biol, 2007,
366,
954-964.
[DOI no: ]
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PubMed id
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Abstract
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Cryptochromes are almost ubiquitous blue-light receptors and act in several
species as central components of the circadian clock. Despite being evolutionary
and structurally related with DNA photolyases, a class of light-driven
DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack
DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs
to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms
resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor
5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to
the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue
crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis
demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the
photoreduction of FAD.
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Figure 1.
Figure 1. (a) Overall structure of Arabidopsis cry3. The
N-terminal antenna domain is shown in green, the FAD-binding
domain in grey. The dimeric organisation is shown on the left.
(b) Structural comparison of A. thaliana cry3 with CryDASH from
S. sp. (magenta, 1NP7), A. thaliana cry1 (orange, 1U3D), E. coli
DNA photolyase (blue, 1DNP) and A. nidulans photolyase (cyan,
1TEZ). The MTHF (orange), FAD (yellow) and 8-HDF chromophores
(blue, from the A. nidulans DNA photolyase) are shown with their
molecular surfaces. The N-terminal extension that is a unique
feature of cry3 is coloured in red. (c) Chromophore arrangement
in the E. coli DNA photolyase. This Figure and Figures 2, 3, 4
were prepared by PyMOL [http://www.pymol.org].
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Figure 4.
Figure 4. The MTHF binding site of A. thaliana cry3. Stereo
diagrams showing the MTHF binding site of (a) A. thaliana cry3
and (c) E. coli DNA photolyase. (b) Schematic diagram of
MTHF–cry3 interactions.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
366,
954-964)
copyright 2007.
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Secondary reference #1
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Title
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Crystallization and preliminary X-Ray analysis of cryptochrome 3 from arabidopsis thaliana.
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Authors
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R.Pokorny,
T.Klar,
L.O.Essen,
A.Batschauer.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2005,
61,
935-938.
[DOI no: ]
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PubMed id
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Figure 1.
(a) Polyacrylamide gel electrophoresis under denaturing
conditions of purified A. thaliana cry3. Lane 1, Sigma
molecular-weight markers 7B (kDa; Sigma-Aldrich, St Louis, MO,
USA). Lane 2, purified cry3 (10 [micro]g) used for
crystallization. The gel was stained with Coomassie blue. (b)
Typical morphology of A. thaliana cry3 crystals. Crystals were
obtained by equilibration against a solution of 85 mM sodium
citrate pH 4.6, 170 mM ammonium acetate, 21.5% PEG 4000 and 7.5%
glycerol. The average dimensions of the cry3 crystals were about
0.2 x 0.1 x 0.1 mm. (c) Self-rotation function calculated with a
native A. thaliana cry3 data set employing data between 20.0 and
2.5 A resolution. A self-rotation search with the program MOLREP
at the angle [chi] = 180[deg] was used to identify the twofold
rotation axis. Acta Crystallogr Sect F Struct Biol Cryst Commun.
2005 October 1; 61(Pt 10): 935–938. Published online 2005
September 30. doi: 10.1107/S1744309105028897. Copyright
[copyright] International Union of Crystallography 2005
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Figure 2.
(a) Absorption spectrum of purified A. thaliana cry3. The
dominant absorption peak at 384 nm typical of MTHF absorption
and the shoulders at 450 and 480 nm typical of absorption of
two-electron oxidized form of FAD are clearly visible. Inset:
absorption spectra in the 500 --700 nm region shown at larger
resolution with two peaks (at 590 and 640 nm) typical of the
absorption of flavin neutral radical FADH . The concentration of
cry3 was 1.3 mg ml^[minus sign]1. (b) Fluorescence emission and
excitation (b) spectra of purified A. thaliana cry3. Emission
spectra are shown as solid lines and excitation spectra as
broken lines. Emission of excited flavin showing a peak at 520
nm and excitation spectra for this emission are shown in red.
Emission of excited MTHF* with a peak at 460 nm overlapping the
flavin emission peak and excitation spectra for emission at 460
nm are shown in blue. Inset: emission and excitation spectra in
the 300 --600 nm region shown at larger resolution. The
concentration of cry3 was 50 [micro]g ml^[minus sign]1. Acta
Crystallogr Sect F Struct Biol Cryst Commun. 2005 October 1;
61(Pt 10): 935–938. Published online 2005 September 30. doi:
10.1107/S1744309105028897. Copyright [copyright] International
Union of Crystallography 2005
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the IUCr
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