Laue Data Research Articles

Towards automated Laue data processing: application to the choice of optimal X-ray spectrum.

Laue data reduction has now reached a level of sophistication that allows nearly automated processing to be performed. The software described enables complete reduction of the data with essentially no user intervention, making Laue processing almost as straightforward as monochromatic data processing. Interactive work is limited to the indexing of only one Laue pattern. More importantly, it is shown that the data quality is substantially enhanced when soft-limited predictions are used. Further improvement obtained by taking advantage of the structure-factor amplitudes from a known closely related structure is described. To determine the most suitable type of insertion device to be used for time-resolved Laue crystallography, the technique described was applied to Laue data sets collected from photoactive yellow protein under identical conditions but with three different insertion devices: a wiggler, a broad-bandpass undulator and a single-line undulator. Although the optimal choice may ultimately be dictated by sample parameters (such as mosaic spread) and by the type of experiment (repeatable or non-repeatable reactions), the results here show that the use of single-line undulators will generally yield by far the best compromise between data quality, acquisition time and radiation damage.

Extraction of accurate structure-factor amplitudes from Laue data: wavelength normalization with wiggler and undulator X-ray sources

Wavelength normalization is an essential part of processing of Laue X-ray diffraction data and is critically important for deriving accurate structure-factor amplitudes. The results of wavelength normalization for Laue data obtained in nanosecond time-resolved experiments at the ID09 beamline at the European Synchrotron Radiation Facility, Grenoble, France, are presented. Several wiggler and undulator insertion devices with complex spectra were used. The results show that even in the most challenging cases, such as wiggler/undulator tandems or single-line undulators, accurate wavelength normalization does not require unusually redundant Laue data and can be accomplished using typical Laue data sets. Single-line undulator spectra derived from Laue data compare well with the measured incident X-ray spectra. Successful wavelength normalization of the undulator data was also confirmed by the observed signal in nanosecond time-resolved experiments. Single-line undulators, which are attractive for time-resolved experiments due to their high peak intensity and low polychromatic background, are compared with wigglers, based on data obtained on the same crystal.

New processing tools for weak and/or spatially overlapped macromolecular diffraction patterns.

Tools originally developed for the treatment of weak and/or spatially overlapped time-resolved Laue patterns were extended to improve the processing of difficult monochromatic data sets. The integration program PrOW allows deconvolution of spatially overlapped spots which are usually rejected by standard packages. By using dynamically adjusted profile-fitting areas, a carefully built library of reference spots and interpolation of reference profiles, this program also provides a more accurate evaluation of weak spots. In addition, by using Wilson statistics, it allows rejection of non-redundant strong outliers such as zingers, which otherwise may badly corrupt the data. A weighting method for optimizing structure-factor amplitude differences, based on Bayesian statistics and originally applied to low signal-to-noise ratio time-resolved Laue data, is also shown to significantly improve other types of subtle amplitude differences, such as anomalous differences.

Accurate and highly complete synchrotron protein crystal Laue diffraction data using the ESRF CCD and the Daresbury Laue software

Developments in electronic area detectors such as CCDs and image plates have transformed the capability of the synchrotron Laue protein crystallography technique compared with film. The rapid readout of CCDs makes practical the use of rather fine angular interval settings of the crystal between each Laue exposure and a large overall angle coverage. The use of the ESRF CCD (image intensifier type) presented here in the Laue data collection on ESRF ID09 (the `Laue beamline') from a single crystal of the 34 kDa wild-type hydroxymethylbilane synthase (HMBS), space group P21212 a = 88.06, b = 75.73, c = 50.35 A, yielded 47 Laue exposures in 2.5° angle intervals from a single crystal. The data processed by the Daresbury Laue software is highly complete (∞–2dmin = 77.5%; 2dmin–dmin= 91.7%) to 2.3 A with high redundancy (11.2). Comparison with calculated structure factors and careful analysis of the Laue geometry shows that between ∞ and 5dmin better completeness still should be possible, which can ideally be realized from CCD detector dynamic range hardware improvements and/or software algorithms to integrate saturated spot profiles. Prospects for Laue diffraction data collection using yet faster detectors such as the `pixel detector' to study irreversible catalytic structural processes in a crystal, the most challenging of all time-resolved experiments, are bright.

Laue crystallography: coming of age

A renewed interest in the Laue diffraction technique has been brought about by the development of new, more intense and brilliant synchrotron sources along with their insertion devices such as wigglers and undulators, and by the prospect of using these sources to study structural dynamics by time-resolved crystallography. Theoretical studies during the past decade have identified unique features of the polychromatic diffraction geometry and greatly improved our understanding of the Laue method. This led to innovative approaches to Laue data processing and its software implementation. Most of the problems in Laue data processing, considered for a long time to limit the applicability of the technique, have been solved. Significant advances have also been made in the development of synchrotron sources, beamline optics and instrumentation, and the X-ray detectors. Static Laue experiments yield structure amplitudes that equal those from monochromatic data in quality. When coupled with careful consideration of data-collection strategies and reaction initiation in crystals, a series of successful time-resolved Laue experiments on biological systems have been conducted. These have revealed information on structural dynamics inaccessible to any other conventional diffraction method. These static and time-resolved experiments demonstrate that the Laue method is coming of age. They also suggest avenues for future improvements: a correct treatment of finite mosaic spread and the associated energy width of Laue spots; consideration of diffuse scattering; and determination of intermediate structures in time-resolved experiments in which those intermediates do not attain a high concentration.

LSCALE– the new normalization, scaling and absorption correction program in the DaresburyLauesoftware suite

A new program in the DaresburyLauesoftware suite has been developed for the scaling and normalization of Laue intensity data, to yield fully corrected structure amplitudes. Previously available routines have been improved, and additional options for refinement, control and statistical diagnostic output provided. A new feature, namely a wavelength- and position-dependent absorption correction that models a two-dimensional surface derived from the Laue data alone, is discussed in detail; it is tested on simulated and real data, and the improvement in data quality is demonstrated. The wavelength normalization function is now able, when sufficiently redundant experimental data are available, to model fine details such as the features arising from the modification of the incident intensity spectrum by a platinum mirror in the beamline optics. A full data set for tetragonal lysozyme is processed with the new program, and extensive statistical output is given.

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.

Estimating unobserved reflection intensities in Laue diffraction by the maximum-entropy method.

In protein crystallography, the use of low-resolution reflections is important in defining the molecular mask and polypeptide backbone. However, in Laue data collection, the loss of low-resolution reflection data (>2dmin) can be as high as 40-50%, even after the deconvolution of multiples. To estimate the reflection intensities that are not recorded in data collection, a new method is presented based on maximizing the entropy of the Patterson function subject to the constraints imposed by the intensities of the observed reflections. The method has been tested with Laue diffraction data from hen egg-white lysozyme. All unobserved reflections within 5 A resolution were estimated, and their inclusion in the electron-density-map calculation significantly improved the connectivity. This method could also be applied to improve the completeness of monochromatic data.

Static Laue diffraction studies on acetylcholinesterase.

Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.

Structure refinement against synchrotron Laue data: strategies for data collection and reduction.

The synchrotron Laue technique has been applied to high-resolution structure refinement of the ribotoxin, restrictocin [Yang & Moffat (1996). Structure, 4, 837-852]. By employing carefully designed data-collection strategies and the data-reduction algorithms incorporated in the software system LaueView [Ren & Moffat (1995a). J. Appl. Cryst. 28, 461-481; Ren & Moffat (1995b). J. Appl. Cryst. 28, 482-493], a set of high-resolution Laue data with a completeness and accuracy comparable to excellent monochromatic data was obtained. Through detailed comparison with the monochromatic data and electron-density maps derived from the Laue data, optimum data-collection and reduction strategies were identified and the application of Laue diffraction techniques to conventional crystallographic refinement was demonstrated.

DAFS measurements using the image-plate Weissenberg method.

An instrumental technique for DAFS measurements which can provide site-specific information is proposed. The approach uses (i) focusing optics with parabolic mirrors and a double-crystal monochromator, (ii) the Laue and Bragg settings and (iii) data collection by the image-plate Weissenberg method. Six image exposures are recorded per plate at five intrinsic energies and one reference energy. The single-crystal measurements were performed at the Co K-absorption edge, and the 200, 220 and 311 reflections of CoO and 511 and 911 reflections of Co(3)O(4) were used for analysis. The regression analysis of chi(k), Fourier transforms of k(3)chi(k) and back-Fourier filtering have been performed.

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 Gd 2O 3-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.

Time-resolved structures of macromolecules at the ESRF: Single-pulse Laue diffraction, stroboscopic data collection and femtosecond flash photolysis

We review the time structure of synchrotron radiation and its use for fast time-resolved diffraction experiments in macromolecular photocycles using flash photolysis to initiate the reaction. The source parameters and optics for ID09 at ESRF are presented together with the phase-locked chopper and femtosecond laser. The chopper can set up a 900 Hz pulse train of 100 ps pulses from the hybrid bunch-mode and, in conjunction with a femtosecond laser, it can be used for stroboscopic data collection with both monochromatic and polychromatic beams. Single-pulse Laue data from cutinase, a 22 kD lipolic enzyme, are presented which show that the quality of single-pulse Laue patterns are sufficient to refine the excited state(s) in a reaction pathway from a known ground state. The flash photolysis technique is discussed and an example is given for heme proteins. The radiation damage from a laser pulse in the femto and picosecond range can be reduced by triggering at a wavelength where the interaction is strong. We propose the use of microcrystals in the range 25–50 μm for efficient photolysis with femto and picosecond pulses. The performance of circular storage rings is compared with the predicted performance of an X-ray free electron laser (XFEL). The combination of micro beams, a gain of 10 5 photons per pulse and an ultrashort pulse length of 100 fs is likely to improve pulsed diffraction data very substantially. It may be used to image coherent nuclear motion at atomic resolution in ultrafast uni-molecular reactions.

Accuracy of Structural Information Obtained at the European Synchrotron Radiation Facility from Very Rapid Laue Data Collection on Macromolecules

The potential of very rapid Laue data collection for time-resolved studies down to the 150 ps timescale has been demonstrated in the case of cutinase, a 22 kDa lipolytic enzyme for which a considerable amount of structural information is available. This paper reports the derivation of the structure of native cutinase at 1.5 Å from a Laue data set recorded at the White Beam Station of the European Synchrotron Radiation Facility (ESRF), with a total exposure time of 8.5 ns. The structure of the heteromorphous mutant R196E was chosen as a starting model for refinement, in order to check whether these fast Laue data were of sufficient quality to allow an accurate structure determination from a strongly biased starting model. This analysis is relevant because similar situations are encountered in fast time-resolved experiments where rapid structural modifications of a protein are analysed from fast Laue data sets, recorded in some excited states of the protein, and from a structural model representative of the rest state. 19 Laue images were recorded with 150 ps X-ray pulses emitted by a single electron bucket from the ESRF storage ring. With two insertion devices used in series, tile available photon flux was sufficient to refine a satisfactory model of native cutinase (Rcryst = 19.3%; Rfree = 24.2%). Discrepancies between this model and an accurate atomic model of cutinase (obtained from monochromatic data collected to 1.0 Å, resolution, Rcryst = 9.7%) were minor and mainly due to the nonoptimal completeness of the data (71.7% to 1.5 Å) and to the different extent in resolution. The wild-type Arg196 could be readily positioned in the electron density and significant main- and side-chain displacements due to packing constraints were successfully retrieved with the Laue data. The electron-density maps were of sufficient quality to solve unambiguously these structural modifications. This feasibility study shows that very rapid Laue diffraction is a powerful tool to study protein dynamics in real time, provided that suitable macromolecular crystals as well as efficient reaction-triggering techniques are available.

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 Åwere 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 Åsynchrotron 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.

Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography.

The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.

Crystal growth of (La,Sr) 2CuO 4 by float zone melting

High quality single crystals of La 2 − x Sr x CuO 4 ( x = 0.15, 0.20) have been successfully grown using the floating zone method. Stoichiometric feed rods were prepared with a density high enough to directly zone refine. The chemical composition of the molten zone was allowed to establish itself during the first few zone lengths, thus no nonstoichiometric seed rod was necessary. Neutron diffraction shows the boule is composed of several large grains. The grain boundaries are found to be Sr-rich, containing La 2SrCu 2O 6. Electron microscopy, X-ray back-reflection Laue and AC susceptibility data are used to characterize the crystals.

Design of the Structural Biology Center beamlines at the APS (abstract)

The Structural Biology Center-CAT will develop and operate a sector of the APS as a user facility for studies in macromolecular crystallography. The techniques applied will include multiple-energy anomalous dispersion (MAD) phasing and polychromatic (Laue) data collection. Data will be recorded on a high resolution CCD-area detector. The SBC is constructing two beamlines, one for radiation from an undulator and one for radiation from a bending magnet. The x ray optics of both beamlines are designed to produce a highly demagnified image of the source in order to match the focal size with the sizes of the sample and the resolution element of the detector. Vertical focusing is achieved by a flat, cylindrically bent mirror. Horizontal focusing is achieved by sagittally bending the second crystal of a double crystal-monochromator. The double-crystal monochromators of both beamlines have a constant exit height output beam. On the undulator beamline, two double-crystal monochromators are installed in series—one with Si-111 crystals and the second with Si-220 crystals—in order to facilitate quick change between high flux and narrow bandwidth. For the heat-loaded first crystals, the liquid-nitrogen-cooled, thin-web design being developed by the APS has been adopted. On the bending magnet beamline, three crystals (Si-111, Si-220, Si-400) are mounted side-by-side on the first crystal stage and translated into the beam is required.

Laue data refinement of the crystal structure of stanfieldite, data collected at CHESS using undulator radiation with image plates in a cylindrical film cassette

Laue data refinement of the crystal structure of stanfieldite, data collected at CHESS using undulator radiation with image plates in a cylindrical film cassette

Insights into specificity of cleavage and mechanism of cell entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin

Background: Restrictocin, a highly specific ribotoxin made by the fungus Aspergillus restrictus, cleaves a single phosphodiester bond in the 28S RNA of eukaryotic ribosomes, inhibiting protein synthesis. The sequence around this cleavage site is a binding site for elongation factors, and is conserved in all cytoplasmic ribosomes. The catalytic mechanism of restrictocin and the reasons for its high substrate specificity are unknown. No structure has been determined for any other member of the Aspergillus ribotoxin family. Results The crystal structure of restrictocin was determined at 2.1 å resolution by single isomorphous replacement and anomalous scattering techniques, and refined to 1.7 å resolution using synchrotron Laue data. The structural core of the protein, in which a three-turn α helix is packed against a five-stranded antiparallel β sheet, can be well aligned with that of ribonuclease T1. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA binding. Conclusion Restrictocin appears to combine the catalytic components of T1 ribonucleases with the base recognition components of Sa ribonucleases. Modeling studies using an NMR structure of an RNA substrate analog suggest that the tertiary structure of the substrate RNA is important in protein–RNA recognition, fitting closely into the concavity of the presumed binding site. We speculate that the large 39-residue loop L3, which has similarities to loops found in lectin sugar-binding domains, may be responsible for restrictocin's ability to cross cell membranes.