Mogens S. Lehmann

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.

On the use of a small two-dimensional position-sensitive detector in neutron diffraction

A small position-sensitive 3He gas detector has been developed for diffraction studies using short-wavelength neutrons. It covers 32 × 32 pixels with a 2 mm resolution, which in the present set up corresponds to 0.25°, and the efficiency is 75% at a wavelength of 0.8 A. The detector has been used for standard data collection, as well as for studies of twinned crystals and measurements involving parasitic reflections. In all cases it has improved the mode and speed of the measurement, with gain factors compared with single detectors which range from more than two in standard measurements to several hundred for the study of details of the Bragg peak(s). The computation time in data analysis and the storage of the data are no major limitations, partly due to the smallness of the data array, and partly due to efficient packing routines.

Statistical Geometry. I. A Self-Consistent Approach to the Crystallographic Inversion Problem Based on Information Theory

The problem of inverting crystallographic diffraction data to obtain structural information is examined within the maximum-entropy formulation of information theory. The principal features of the present method (termed statistical geometry) are: (i) all predictions of the method are consistent with the given information (constraints) and least biased with respect to missing information, (ii) the adoption of weak (typically non-linear) constraints for incorporating the major part of the structural information guarantees that a solution exists in practice and leads to filtering of the structure maps consistent with the accuracy of the data, (iii) general conditions are established which lead to unique solutions for the structure map, (iv) atomicity is not a prerequisite, (v) other methods of crystallographic inversion may be incorporated via the adoption of appropriate constraint relations, and (vi) the task of numerical solution is roughly linear in the number of reflexions and in the number of pixels in the structure, and involves only straightforward numerical techniques. These features suggest that the method is especially well suited to problems such as the structure determination of biological macromolecules, and the determination of high-resolution electron-density maps, although it manifestly provides a general framework for treating a wide class of image-processing problems.

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.

Rapid neutron-diffraction data collection for hydrogen-bonding studies: application of the Laue diffractometer (LADI) to the case study zinc (tris)thiourea sulfate

The successful application of the newly developed image-plate neutron Laue diffractometer (LADI) at the Institut Laue-Langevin (ILL), Grenoble, France, for rapid hydrogen-bonding characterization is reported. The case study concerns the promising non-linear optical material zinc (tris)thiourea sulfate (ZTS), which contains 30 atoms in the asymmetric unit and crystallizes in the orthorhombic space group, Pca2(1), a = 11.0616 (9), b = 7.7264 (6), c = 15.558 (1) A [T = 100.0 (1) K]. The results from a 12 h data collection from ZTS on LADI are compared with those obtained over 135 h using the monochromatic four-circle diffractometer D9 at the same reactor source with a crystal 13 times larger in volume. Both studies reveal the extensive hydrogen bonding and other close non-bonded contacts within the material. As expected, the results from D9 are more precise than those obtained from LADI; however, the bond geometry determined from the two experiments is the same within the larger estimated standard deviations. Furthermore, the conclusions drawn from the two studies separately regarding the nature of all supramolecular features are identical. This illustrates that LADI is eminently suitable for rapid characterization of hydrogen-bonded structures by neutron diffraction, with the gain in speed compared with traditional instrumentation being several orders of magnitude.

Characterization of image plates for neutron diffraction

A method to measure the overall gain and point-spread function of an image-plate detector system (converter, phosphor, readout electronics) online from single-crystal diffraction patterns is derived. Only with a knowledge of these quantities can one estimate without bias the true standard deviations of the observed integrated intensities of Bragg reflections measured with such a detector. The gain corrected for the point-spread function should ideally be approximately 1.0 to avoid loss of data or precision due to the upper and lower limits of digitization of the diffraction patterns. The gain and the point-spread function are derived for several configurations of the three neutron Laue image-plate diffractometers, LADI, VIVALDI and LADI-III, at the Institut Laue–Langevin. The detective quantum efficiencies (DQE) of these instruments have been measured to be 0.16 (1), 0.57 (2) and 0.46 (3), respectively. It has also been found that the DQE is effectively constant for different times of exposure, despite the fading of the photostimulated luminescent signal during that time. While the results described in this paper concern neutron detection by image plates, the method is equally valid for other integrating detectors and radiations, e.g. CCD detectors and X-rays.

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.