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At the Institut Laue-Langevin a newly upgraded neutron Laue diffractometer, LADI-III, has been operational since March 2007. The instrument uses a large cylindrical area detector composed of neutron-sensitive image-plates (NIPs) which completely surround the sample and allows large numbers of Bragg reflections to be recorded simultaneously. A detector employing NIPs is comparatively cheap, is capable of high spatial resolution, has good homogeneity, a large dynamical range, extended linearity and no dead-time, and it can be made to subtend very large angles at the specimen. The neutron-sensitive plates are based on the same storage-phosphor (BaFBr doped with Eu2+ ions) which is used for X-ray image plates, but with Gd2O3 added. This enables the Gd nuclei to act as neutron scintillators by creating a cascade of γ-rays and conversion electrons. Data is collected using a quasi-Laue method in order to provide a rapid survey of reciprocal space, while reducing the background on the detector compared to use of the full white beam. Various Ni/Ti multilayer filters are available so as to select the wavelength range (δλ/λ) and wavelength (λ) that is best suited to the sample (Høghøj P., et al., 1996, J Phys Soc Jpn. 65:296-2983). The sample crystal is mounted on a goniometer head on the cylinder axis, and can be rotated around this axis. The neutron beam, which enters and leaves via opposed holes in the cylinder, produces Bragg reflections which are recorded on the image plates mounted on the inside cylindrical surface.
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An improved read/erase system has been incorporated so that a miniaturized reading head located inside the drum scans the image-plate. The detector, which has a radius of 200mm and a length of 450mm is read off in a phonographic mode with the reading head tracking slowly vertically while the cylinder rotates at high speed (500rpm). The readout time is ~5 minutes, giving an image of the pattern recorded on the plates comprising 4980 x 1800 square pixels 250µm on edge.
Comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, indicate that the transferal of the image-plates and readout system internally provides a 2- to 3-fold gain in neutron detection (Wilkinson C, Blakeley MP, Dauvergne F: ILL internal report, 2007, ILL07WI02T) allowing data collection to higher resolution (~1.5 Å), using shorter exposure times and smaller crystal volumes. The improved neutron detector efficiency of LADI-III coupled with the use of perdeuterated biological samples has enabled neutron protein crystallography to become more accessible to the structural biology community, extending the size and complexity of systems that can be studied (~150 Å on cell edge) while lowering the sample volumes required (~0.1- 0.2mm3).
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 - The new Laue diffractometer 'LADI-III' installed on cold guide H142 at the Institut Laue-Langevin. Key; 1 = sample holder, 2 = cylindrical detector, 3 = internal read/erase system, 4 = neutron image-plates. The improved neutron detection efficiency of LADI-III (relative to LADI-I) enables us to use much smaller crystal volumes (~0.1 - 0.2mm3) and/or study larger unit-cell systems (~150 Å on cell edge). Data can also be collected with reduced exposure times and/or to higher resolution.
Guide hall n°1, cold guide H142 | Monochromators | Quasi-Laue (δλ/λ≤25%) | Ni/Ti multilayer band-pass filter | Various multilayer band-pass filters (λ=3.5 Å) | δλ/λ from 5 to 25% | Collimation | Pinholes | 0.1 to 2.9 mm | Flux at specimen | (λ = 3.5 Å; δλ/λ = 20%) | 3 x 107 n cm-2 s-1 | Detector | Cylinder with internal neutron image plates and read/erase system | Neutron image plate | Gd2O3 doped BaF(Br.I):Eu2+ | Radius | 200 mm | Length | 450 mm | Active area | 1245 x 450 mm2 | Angle subtended | 172° in θ, 49° in ν | Pixel size | 125, 250, 500 µm2 | Sample environment | Ambient or displex cryostat going to approx. 15 K. |
 - Photo of a perdeuterated human aldose reductase crystal in complex with NADP+ and the inhibitor IDD594. The crystal volume is approximately 0.15 mm3 and neutron Laue data were collected to 2.2 Å on the LADI-I instrument. The protein crystallizes in space group P21 with unit-cell constants a = 49.3 Å, b = 66.6 Å and c = 47.3 Å, α = 90.0°, β = 92.4°, γ = 90.0°.
A key advance for the development of neutron macromolecular crystallography is the ability to produce fully deuterated single crystals, in which all H-atoms are replaced by D-atoms. Hydrogen has a large incoherent cross section and as hydrogen atoms account for about 50% of the atoms in a protein and are also present in the surrounding water solvent molecules, the signal-to-noise ratio of neutron diffraction data from fully hydrogenated systems is dominated by the large incoherent background. In contrast, the incoherent scattering arising from deuterium is 40 times lower, whilst the neutron scattering length is positive and twice that of hydrogen. Therefore, replacing hydrogen by deuterium in protein crystals both increases the coherent scattering signal and decreases the incoherent background. Fully deuterated samples are produced by expressing the protein under deuterated conditions in vivo and enable us to use radically smaller crystals for neutron data collection. Clearly the volume of the crystal necessary is dependant on the unit-cell and space group of the particular system being studied, however, crystals with volumes of 0.1 - 0.2mm3 are now providing high resolution data. This was demonstrated for the case of human aldose reductase (36kDa) for which neutron data to 2.2 Å resolution were collected on LADI-I from a perdeuterated aldose reductase crystal with volume of only 0.15 mm3 (1, 2). Since the growth of very large single crystals is the major bottleneck in neutron protein crystallography, this result demonstrates the critical advance offered by perdeuteration.
The ILL, in collaboration with the EMBL-Grenoble Outstation, was the first to create a Deuteration Laboratory (D-Lab) dedicated to the requirements of neutron scattering. The basis of the original proposal was that biological neutron scattering studies greatly underexploited their potential simply because scientists had no easy access to the continuity of expertise and facilities that were necessary to allow efficient and cost-effective labelling of biological molecules. The ILL management and its peer review committees recognised that major developments in instrumentation should be paralleled by adequate investment in laboratories for deuteration.The ILL-EMBL Deuteration Laboratory has been running since 2003 as a user facility and reached the final stage of its development when it moved into the Partnership for Structural Biology in 2006. The laboratory has a thriving user programme that is accessible by the user community through a peer-review process (directly analogous to the ILL beam time proposal system). The laboratory is also involved in the development of new approaches that are steadily extending the scope of labeling methods (3, 4) and has benefited from synergy with the solid state and solution NMR communities.
References
1. Hazemann, I., Dauvergne, M.T., Blakeley, M. P., Meilleur, F., Haertlein, M., Van Dorsselaer, A., Mitschler, A., Myles, D. A. A. & Podjarny, A. D. (2005) Acta Cryst. D61, 1413-1417.
2. Blakeley, M. P., Ruiz, F., Cachau, R., Hazemann, I., Meilleur, F., Mitschler, A., Ginell, S., Afonine, P., Ventura, O. N., Cousido-Siah, A., Haertlein, M., Joachimiak, A., Myles, D. A. A. & Podjarny, A. D. (2008) Proceedings Natiaonal Academy of Sciences, USA. 105(6), 1844-1848.
3. Callow, P., Sukhodub, A., Taylor, J. E. & Kneale, G. G. (2007) Journal of Molecular Biology 369, 177-185.
4. Tehei, M., Franzetti, B., Wood, K., Gabel, F., Fabiani, E., Jasnin, M., Zamponi, M., Oesterhelt, D., Zaccai, G., Ginzburg, M. & Ginzburg, B. Z. (2007) Proceedings of the National Academy of Sciences, USA 104, 766-771.
Obtaining deuterated protein crystals suitable for neutron analysis implies a systematic study of the solubility and protein-protein interactions in deuterated crystallization conditions as H-D exchange alters the physico-chemical properties of protein solutions and affects the crystallization process in a significant way. We developed a novel method and apparatus that finds application in the growth of large high-quality crystals for neutron crystallography (1 - 3). It allows the manipulation of the kinetics of the crystallization process, taking advantage of generic features of the phase diagram. The promotion of crystal growth is obtained by keeping the crystallization solution metastable during the process of crystal growth. This is achieved by regulating the temperature of the crystallization solution using control parameters determined in situ during the growth process. Knowledge of the phase diagram and the ability to control the temperature to drive the process of the crystallization of biomacromolecules allow us to tailor crystallization experiments to search for conditions that lead to crystals of a desired crystalline form, quality and size. The technique has been used to grow large crystals for neutron diffraction experiments of several proteins of interest such as rat g-crystallin E, PA-IIL lectin from Pseudomonas aeruginosa, yeast inorganic pyrophosphatase, urate oxidase from Aspergillus flavus and human carbonic anhydrase II.
References
1. Budayova-Spano, M., Dauvergne, F., Audiffren, M., Bactivelane, T. & Cusack, S. (2007) Acta Cryst. D63(Pt 3):339-47.
2. Budayova-Spano, M., Fisher, S. Z., Dauvergne, M. T., Agbandje-McKenna, M., Silverman, D. N., Myles, D. A. A. & McKenna, R. Acta Cryst. F62(Pt 1):6-9.
3. Budayova-Spano, M., Bonneté, F., Ferté, N., El Hajji, M., Meilleur, F., Blakeley, M. P. & Castro, B. (2006) Acta Cryst. F62, 306-309
 - Phase diagram illustrating the control process for crystal growth in the metastable zone in the case of a protein with direct solubility (solubility increases with the temperature). The solubility curve (red points) as well as the sequence representing the various steps (blue arrows) of the actual crystal-growth process of hydrogenated recombinant urate oxidase complexed with 8-azaxanthin in the presence of D2O is schematized. The photographs show the crystal habits and volume of a seeded crystal observed before equilibration at 298, 293, 288, 283 and 278 K, respectively.
 - An instrument for the temperature-controlled optimization of crystal growth.
 - Close-up of a neutron Laue diffraction pattern from a large cryo-cooled crystal of rubredoxin (from the archaebacterium Pyrococcus furiosus) at 15K. The volume of the cryo-cooled crystal was 1.4 mm3.
As neutrons do not cause any observable radiation damage in protein crystals neutron data is generally collected at room temperature. However, cryo-cooling protein crystals in X-ray crystallography has been shown to provide potential improvements in data quality by reducing B-factors, and perhaps more interestingly opens up the possibility for freeze-trapping studies of enzymatic reaction intermediates (Moffat K., 1995, Biotechnology). Data collection at cryo-temperatures is a challenge when using the large protein crystals used for neutron protein crystallography since the solvent must be rapidly (flash)-cooled to a vitreous glass in order to avoid ice formation that disrupts the crystal lattice. Protocols for cryo neutron crystallography have been developed at the ILL/EMBL to cool and maintain large protein crystals (1 - 5mm3) at cryogenic temperatures (~15K). Data have been collected at 15K on LADI-I for lysozyme (Myles D., 2003, ACA Transactions 38; Meilleur F., et al., 2005, Neutron Laue analysis of hydrogen and hydration in protein structure. 'Hydrogen and hydration sensitive structural biology'. p75-85), concanavalin A (Blakeley M., et al., 2004 Proc Natl Acad Sci USA 101(47):16405-16410) and rubredoxin (Blakeley M., et al., 2008, in preparation). These results open up new experimental capabilities that will allow analysis of structure (and transitions) as a function of temperature and the ability to use freeze trapping to 'quench' kinetic processes and capture reaction intermediaries, allowing new scientific questions to be addressed. Several projects planned for data collection on LADI-III in 2008 aim to address such questions.
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