C. Wilkinson

Neutron Laue diffractometry with an imaging plate provides an effective data collection regime for neutron protein crystallography.

Neutron quasi-Laue diffraction data (2 Å resolution) from tetragonal hen egg-white lysozyme were collected in ten days with neutron imaging plates. The data processing Laue software, LAUEGEN, developed for X-ray Laue diffractometry, was adapted for neutron diffractometry with a cylindrical detector. The data analysis software, X-PLOR, was modified and used for the refinement of hydrogen atoms, and the positions of 960 hydrogen atoms in the protein and 157 bound water molecules, were determined. Several examples are given of the methods used to identify hydrogen atoms and water molecules.

Quasi-Laue neutron-diffraction study of the water arrangement in crystals of triclinic hen egg-white lysozyme.

Triclinic crystals of lysozyme, hydrogen-deuterium exchanged in deuterated solvent, have been studied using neutron quasi-Laue techniques and a newly developed cylinder image-plate detector. The wavelength range employed was from 2.7 to 3.5 A, which gave 9426 significant reflections [F >/= 2sigma(F)] to a resolution limit of 1. 7 A. The deuteration states of the H atoms in the protein molecule were identified, followed by an extensive analysis of the water structure surrounding the protein. The final R factor was 20.4% (Rfree = 22.1%). In total, the 244 observed water molecules form approximately one layer of water around the protein with far fewer water molecules located further away. Water molecules covering the apolar patches make tangential layers at 4-5 A from the surface or form C-H...O contacts, and several water-molecule sites can be identified in the apolar cavities. Many of the water molecules are apparently orientationally disordered, and only 115 out of the 244 water molecules sit in mean single orientations. Comparison of these results with quasi-elastic neutron scattering observations of the water dynamics leads to a picture of the water molecules forming an extended constantly fluctuating network covering the protein surface.

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050.

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.

Direct determination of the positions of the deuterium atoms of the bound water in ­concanavalin A by neutron Laue crystallography

The correct positions of the deuterium (D) atoms of many of the bound waters in the protein concanavalin A are revealed by neutron Laue diffraction. The approach includes cases where these water D atoms show enough mobility to render them invisible even to ultra-high resolution synchrotron-radiation X-ray crystallography. The positions of the bound water H atoms calculated on the basis of chemical and energetic considerations are often incorrect. The D-atom positions for the water molecules in the Mn-, Ca- and sugar-binding sites of concanavalin A are described in detail.

Neutron Laue diffraction study of concanavalin A The proton of Asp28

Neutron Laue data collection, which harnesses a broader wavelength band emitted from the neutron source, opens up the prospect of studying larger proteins and/or using smaller protein crystals than is possible with monochromatic neutron protein crystallography data collection methods. Concanavalin A, a 25 kDa† protein was used in this study, albeit with a rather large crystal of 1.2×1.8×2.2 mm. Data in a resolution range of 8–2.75 Awere used to refine the protein structure, which included many H/D sites; the final R-factor for the protein model and 61 waters was 0.207 (Rfree = 0.310) for 4909 unique reflections. In particular, for example, the proton on Asp28 of concanavalin A, located previously by our 0.94 Asynchrotron X-ray study, was also found in this neutron study; thus the two methods confirm each other. The Asp28 proton was found not to exchange, under the deuteriation conditions used. Negative neutron density was also observed for the manganese binding site consistent with the negative neutron scattering factor for this element. Concanavalin A is one of the first proteins studied by the neutron Laue technique. The limited exchange of H for D almost certainly can be improved upon thus reducing the proton background in the diffracton pattern. This in turn would allow the weaker, high-resolution reflections, to be recorded.

Neutron Laue diffraction in macromolecular crystallography

The time scales required for conventional neutron diffraction analysis of biological single crystals at, or near, atomic resolution are prohibitive - such studies are rarely performed. Laue (white beam) diffraction can provide a more rapid and efficient survey of reciprocal space, maximising the flux at the sample and stimulating large numbers of reflections simultaneously. A LAue DIffractometer (LADI), designed specifically for macromolecular crystallography, has been installed on a cold neutron guide at ILL. The detector comprises a large Gd2O3-doped neutron-sensitive image plate (400 × 800 mm) mounted on a cylindrical camera (318 mm diameter) that is read in phonographic mode after exposure. Detector response has been evaluated and performance indicators are given. Narrow (Quasi-Laue) band-passes (d/gl/λ = 8–20%) are often required for large unit-cell biological crystals in order to reduce reflection overlap and incoherent background. Laue and Quasi-Laue data have now been collected for a number of proteins and other biological crystals. Recent results are presented and future prospects reviewed.

A large image-plate detector for neutrons

Neutron-diffraction studies are much improved by the use of large position-sensitive detectors. Such devices can be based on image-plate technology, which relies on re-usable photostimuable phosphors, combined with a neutron to γ-ray converter. A cylindrical detector has been built, and a first recording of a neutron-diffraction pattern from a protein crystal is presented.

THE OPTIMIZATION OF THE NEUTRON SENSITIVITY OF IMAGE PLATES

Abstract Image plates containing varying concentrations of the neutron converter Gd 2 O 3 and the storage phosphor BaFBr:Eu 2+ have been prepared. Their neutron sensitivity was measured for a neutron wavelength of 3 A and compared with a new model, which is in good agreement with the measured data, showing that the sensitivity, which is the photostimulated light output per irradiated neutron, of image plates is always higher if the neutron irradiation and the readout are done from the same side. In this case an optimum in sensitivity can be obtained for volume fractions of Gd 2 O 3 between 20% and 30% of the total volume of storage phosphor and converter in the material. In the case of neutron irradiation and readout from opposite sides the volume fraction of Gd 2 O 3 should be varied reciprocally with the thickness d of the phosphor layer in order to obtain a maximum of sensitivity. The experimental results and the model indicate that the maximum sensitivity is lower by a factor between 2 and 4 compared with the readout from the same side. The relation between sensitivity and detective quantum efficiency is discussed.

Neutron detection with imaging plates Part I. Image storage and readout

Abstract The detection of neutrons with imaging plates is based on a sequence of physical processes, which are happening during the neutron exposure and the readout of the image information. These processes are investigated in detail starting with the neutron absorption and conversion to secondary radiation by various neutron converter materials and ending with the detection of the photostimulated luminescence, which is emitted during the readout. It is shown that these processes can be quantified by several key parameters, such as i.e. the neutron absorption cross section, the emitted secondary energy and the conversion efficiency of released secondary energy to storage centers in the storage phosphor. The resulting detector characteristics are described in the second part, namely, Neutron detection with imaging plates – part II: detector characteristics [Nucl. Instr. and Meth. A 424 (1999) 34–39].

Neutron Laue diffraction studies of coenzyme cob(II)alamin

Using a recently designed neutron single-crystal diffractometer utilizing a narrow-band Laue concept (LADI), diffraction data were collected from a crystal of the coenzyme cob(II)alamin (B12r), crystallized from a mixture of D2O and perdeuterated acetone. The instrument was placed at the end of a cold neutron guide at the Institute Laue Langevin (ILL, Grenoble, France), and data collection with neutrons of 1.8-8.0 A wavelength to a crystallographic resolution of 1.43 A was complete after about 36 h. This compares favourably with a previous experiment utilizing the same crystal specimen, where more than four weeks were required to collect monochromatic diffraction data to about 1 A resolution. Using the Laue data, the structure was solved by molecular replacement with the known X-ray crystal structure. Difference density maps revealed the atomic positions (including deuterium atoms) of seven ordered solvent water molecules and two (partially disordered) acetone molecules. These density maps were compared with corresponding maps computed with monochromatic neutron-diffraction data collected to 1. 0 A resolution using the same crystal specimen, as well as to maps derived from high-resolution (0.90 A) synchrotron X-ray data. In spite of the better definition of atomic positions in the two high-resolution maps, the 1.43 A LADI maps show considerable power for the determination of the location of hydrogen and deuterium positions.