PDBsum entry 2c8q

Hormone

PDB id: 2c8q

Contents

Protein chains

21 a.a.

29 a.a.

Waters ×31

PDB id:

2c8q

Name:

Hormone

Title:

Insuline(1sec) and uv laser excited fluorescence

Structure:

Insulin a chain. Chain: a. Insulin b chain. Chain: b

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: pancreas. Organ: pancreas

Biol. unit:

Tetramer (from PDB file)

Resolution:

1.95Å R-factor: 0.208 R-free: 0.230

Authors:

X.Vernede,B.Lavault,J.Ohana,D.Nurizzo,J.Joly,L.Jacquamet,F.Felisaz, F.Cipriani,D.Bourgeois

Key ref:

X.Vernede et al. (2006). UV laser-excited fluorescence as a tool for the visualization of protein crystals mounted in loops. Acta Crystallogr D Biol Crystallogr, 62, 253-261. PubMed id: 16510972 DOI: 10.1107/S0907444905041429

Date:

06-Dec-05 Release date: 08-Mar-06

Protein chain

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 21 a.a.

Protein chain

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 29 a.a.

DOI no: 10.1107/S0907444905041429

Acta Crystallogr D Biol Crystallogr 62:253-261 (2006)

PubMed id: 16510972

UV laser-excited fluorescence as a tool for the visualization of protein crystals mounted in loops.

X.Vernede, B.Lavault, J.Ohana, D.Nurizzo, J.Joly, L.Jacquamet, F.Felisaz, F.Cipriani, D.Bourgeois.

ABSTRACT

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.

Selected figure(s)

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.

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.]).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2006, 62, 253-261) copyright 2006.

Figures were selected by the author.