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PDBsum entry 2c8q
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References listed in PDB file
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Key reference
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Title
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Uv laser-Excited fluorescence as a tool for the visualization of protein crystals mounted in loops.
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Authors
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X.Vernede,
B.Lavault,
J.Ohana,
D.Nurizzo,
J.Joly,
L.Jacquamet,
F.Felisaz,
F.Cipriani,
D.Bourgeois.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2006,
62,
253-261.
[DOI no: ]
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PubMed id
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Abstract
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Structural proteomics has promoted the rapid development of automated protein
structure determination using X-ray crystallography. Robotics are now routinely
used along the pipeline from genes to protein structures. However, a bottleneck
still remains. At synchrotron beamlines, the success rate of automated sample
alignment along the X-ray beam is limited by difficulties in visualization of
protein crystals, especially when they are small and embedded in mother liquor.
Despite considerable improvement in optical microscopes, the use of visible
light transmitted or reflected by the sample may result in poor or misleading
contrast. Here, the endogenous fluorescence from aromatic amino acids has been
used to identify even tiny or weakly fluorescent crystals with a high success
rate. The use of a compact laser at 266 nm in combination with non-fluorescent
sample holders provides an efficient solution to collect high-contrast
fluorescence images in a few milliseconds and using standard camera optics. The
best image quality was obtained with direct illumination through a viewing
system coaxial with the UV beam. Crystallographic data suggest that the employed
UV exposures do not generate detectable structural damage.
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Figure 4.
Figure 4 Fluorescence images recorded with the standard setup. A
crystal of cephamycinase 2 ( [127]~ 20 µm in thickness) is
shown in three different orientations in visible light (a) or UV
light (b). Red points show the crystal centre as detected by the
C3D software. The crystal is hardly detectable in visible light,
so that C3D fails to identify it correctly in orientations 2 and
3. In contrast, the crystal is easily identified under UV-laser
illumination by both the user and the software, whatever the
loop orientation.
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Figure 7.
Figure 7 Effect of UV-induced radiation damage. Experimental
difference electron-density maps (F[obs-UV] - F[obs-noUV]) are
shown for insulin. Maps are contoured at ±5.0  ,
where is
the standard deviation of the electron-density difference (red,
negative; green, positive) and are overlaid on a model of
non-irradiated insulin. (a) Overall view of the protein for a 1
s exposure to 266 nm laser light (the same difference map
displayed at ±3.0 is
also featureless and shows only noise peaks). (b) The same view
of the protein for a 60 s exposure to 266 nm laser light (with
identical power density as in a). (c) Example of a damaged
disulfide bridge. Breakage of the CysA7-CysB7 bridge is clearly
visible, together with a significant displacement of the
main-chain carbonyl group of CysA7. The maps were calculated
using CCP4 (Collaborative Computational Project, Number 4,
1994[Collaborative Computational Project, Number 4 (1994). Acta
Cryst. D50, 760-763.]). This figure was drawn using BOBSCRIPT
(Esnouf, 1999[Esnouf, R. M. (1999). Acta Cryst. D55, 938-940.])
and RASTER3D (Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D.
J. (1997). Methods Enzymol. 277, 505-524.]).
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2006,
62,
253-261)
copyright 2006.
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