Laue Data Research Articles

Scaling and Merging Time-Resolved Laue Data with Variational Inference.

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

Pattern-matching indexing of Laue and monochromatic serial crystallography data for applications in materials science.

Serial crystallography data can be challenging to index, as each frame is processed individually, rather than being processed as a whole like in conventional X-ray single-crystal crystallography. An algorithm has been developed to index still diffraction patterns arising from small-unit-cell samples. The algorithm is based on the matching of reciprocal-lattice vector pairs, as developed for Laue microdiffraction data indexing, combined with three-dimensional pattern matching using a nearest-neighbors approach. As a result, large-bandpass data (e.g. 5-24 keV energy range) and monochromatic data can be processed, the main requirement being prior knowledge of the unit cell. Angles calculated in the vicinity of a few theoretical and experimental reciprocal-lattice vectors are compared, and only vectors with the highest number of common angles are selected as candidates to obtain the orientation matrix. Global matching on the entire pattern is then checked. Four indexing options are available, two for the ranking of the theoretical reciprocal-lattice vectors and two for reducing the number of possible candidates. The algorithm has been used to index several data sets collected under different experimental conditions on a series of model samples. Knowing the crystallographic structure of the sample and using this information to rank the theoretical reflections based on the structure factors helps the indexing of large-bandpass data for the largest-unit-cell samples. For small-bandpass data, shortening the candidate list to determine the orientation matrix should be based on matching pairs of reciprocal-lattice vectors instead of triplet matching.

Seed-skewness algorithm for X-ray diffraction signal detection in time-resolved synchrotron Laue photocrystallography.

A one-dimensional seed-skewness algorithm adapted for X-ray diffraction signal detection is presented and discussed. The method, primarily designed for photocrystallographic time-resolved Laue data processing, was shown to work well for the type of data collected at the Advanced Photon Source and European Synchrotron Radiation Facility. Nevertheless, it is also applicable inthe case of standard single-crystal X-ray diffraction data. The reported algorithm enables reasonable separation of signal from the background in single one-dimensional data vectors as well as the capability to determine small changes of reflection shapes and intensities resulting from exposure of the sample to laser light. Otherwise, the procedure is objective, and relies only on skewness computation and its subsequent minimization. The new algorithm was proved to yield comparable results to the Kruskal-Wallis test method [Kalinowski, J. A. et al. (2012). J. Synchrotron Rad. 19, 637], while the processing takes a similar amount of time. Importantly, in contrast to the Kruskal-Wallis test, the reported seed-skewness approach does not need redundant input data, which allows for faster data collections and wider applications. Furthermore, as far as the structure refinement is concerned, the reported algorithm leads to the excited-state geometry closest to the one modelled using the quantum-mechanics/molecular-mechanics approach reported previously [Jarzembska, K.N. et al. (2014). Inorg. Chem. 53, 10594], when the t and s algorithm parameters are set to the recommended values of 0.2 and 3.0, respectively.

LaueG software for displaying and processing neutron Laue images

An overview is presented of an integrated software package for the processing of Laue data from reactor and other continuous neutron sources. LaueG appears to the user as a graphical user interface with interactive graphic modes that depend on the task being performed: simple image display, cell indexing and refinement, peak integration, wavelength normalization and intensity corrections, or identification of statistical outliers. Other capabilities under development, though still usable, include satellite spots and modulated structures, twinned crystal analysis, and ab initio indexing of unit cells. The emphasis is on accurate intensities for structure refinement using an integrated graphical approach.

X-ray micro Laue diffraction tomography analysis of a solid oxide fuel cell.

The relevance of micro Laue diffraction tomography (µ-LT) to investigate heterogeneous polycrystalline materials has been studied. For this purpose, a multiphase solid oxide fuel cell (SOFC) electrode composite made of yttria-stabilized zirconia and nickel oxide phases, with grains of about a few micrometres in size, has been analyzed. In order to calibrate the Laue data and to test the technique's sensitivity limits, a monocrystalline germanium sample of about 8 × 4 µm in cross-section size has also been studied through µ-LT. The SOFC and germanium Laue diffraction pattern analyses are compared and discussed. The indexing procedure has been successfully applied for the analysis of the germanium Laue data, and the depth-resolved two-dimensional cartographies of the full deviatoric strain tensor components were obtained. The development and application of an original geometrical approach to analyze the SOFC Laue data allowed the authors to resolve grains with sizes of about 3 µm and to identify their individual Laue patterns; by indexing those Laue patterns, the crystalline phases and orientations of most of the grains identified through the geometrical approach could be resolved.

In situ serial Laue diffraction on a microfluidic crystallization device.

Renewed interest in room-temperature diffraction has been prompted by the desire to observe structural dynamics of proteins as they function. Serial crystallography, an experimental strategy that aggregates small pieces of data from a large uniform pool of crystals, has been demonstrated at synchrotrons and X-ray free-electron lasers. This work utilizes a microfluidic crystallization platform for serial Laue diffraction from macroscopic crystals and proposes that a collection of small slices of Laue data from many individual crystals is a realistic solution to the difficulties in dynamic studies of irreversible biochemical reactions.

On the assessment of time-resolved diffraction results

Dynamic-structure crystallography, and in particular photocrystallography, is a field of great interest because of its numerous applications. The field covers the studies of reactions in crystals including non-reversible reactions, phase transitions and the structure change on excitation leading to short-lifetime species. Data collected during dynamic-structure crystallography experiments require appropriate tools to perform data analysis. The relative scaling of different sets of data recurs in time-resolved pump-probe data collection in which crystals often disintegrate before a full set of data can be collected. We discuss relative scaling and its use with the Ratio method [1], in which ratios of observed intensities collected with and without light-exposure are used as observables. The scaling method has been developed specifically for pump-probe Laue data, but is not limited to polychromatic data sets. The visualization of the externally induced structure changes in the crystal environment is of importance to check the presence of a structural response (see Fig. a) and hence to define a starting point for the structure refinement. For this purpose, ratio-based Fourier difference maps have been defined. After the structure refinement using the software LASER [2], the quality of the light-exposed structure model can be checked in direct space by visualizing the ratio-based Fourier residual maps, and, in a complementary manner, in reciprocal space by performing statistical analysis on the least-squares (LS) residual vector components with respect to the corresponding H-vectors (see Fig. b). The latter allows detection of any outliers or unexpected tendencies such as dependence of the residual vector components on the resolution or the angular direction in the reciprocal space. Application to the complex [Cu(I)-(1,10-phenanthroline-N,N′)bis(triphenylphosphine)][BF4] will be presented [3]. Research supported by the National Science Foundation (CHE1213223).

Energy resolution of the CdTe-XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals

The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL. The X-ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging-plate detector and then measured with both the XPAD3S-CdTe and the XPAD3S-Si, a silicon photon-counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit-cell parameter determination and the measurement of high-quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X-ray diffraction opened up by this type of technology are discussed.

The kinetic dose limit in room-temperature time-resolved macromolecular crystallography.

Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.

PH Dependence of the Photoactive Yellow Protein Photocycle Investigated by Time-Resolved Crystallography

Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values.

TheLaueUtiltoolkit for Laue photocrystallography. I. Rapid orientation matrix determination for intermediate-size-unit-cell Laue data

A new method for determination of the orientation matrix of Laue X-ray data is presented. The method is based on matching of the experimental patterns of central reciprocal lattice rows projected on a unit sphere centered on the origin of the reciprocal lattice with the corresponding pattern of a monochromatic data set on the same material. This technique is applied to the complete data set and thus eliminates problems often encountered when single frames with a limited number of peaks are to be used for orientation matrix determination. Application of the method to a series of Laue data sets on organometallic crystals is described. The corresponding program is available under a Mozilla Public License-like open-source license.

Neutron structure of type‐III antifreeze protein allows the reconstruction of AFP–ice interface

Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.

Time-resolved synchrotron diffraction and theoretical studies of very short-lived photo-induced molecular species

Definitive experimental results on the geometry of fleeting species are at the time of writing still limited to monochromatic data collection, but methods for modifications of the polychromatic Laue data to increase their accuracy and their suitability for pump-probe experiments have been implemented and are reviewed. In the monochromatic experiments summarized, excited-state conversion percentages are small when neat crystals are used, but are higher when photoactive species are embedded in an inert framework in supramolecular crystals. With polychromatic techniques and increasing source brightness, smaller samples down to tenths of a micrometre or less can be used, increasing homogeneity of exposure and the fractional population of the excited species. Experiments described include a series of transition metal complexes and a fully organic example involving excimer formation. In the final section, experimental findings are compared with those from theoretical calculations on the isolated species. Qualitative agreement is generally obtained, but the theoretical results are strongly dependent on the details of the calculation, indicating the need for further systematic analysis.

Feasibility of one-shot-per-crystal structure determination using Laue diffraction.

Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a 'pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20-30 microm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase.

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 A, 100K; 0.80 A, 15K; 1.75 A, 293K), neutron Laue data (2.2 A, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of approximately 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Analysis of experimental time-resolved crystallographic data by singular value decomposition.

Singular value decomposition (SVD) separates time-dependent crystallographic data into time-independent and time-dependent components. Procedures for the effective application of SVD to time-resolved macromolecular crystallographic data have yet to be explored systematically. Here, the applicability of SVD to experimental crystallographic data is tested by analyzing 30 time-resolved Laue data sets spanning a time range of nanoseconds to milliseconds through the photocycle of the E46Q mutant of photoactive yellow protein. The data contain random and substantial systematic errors, the latter largely arising from crystal-to-crystal variation. The signal-to-noise ratio of weighted difference electron-density maps is significantly improved by the SVD flattening procedure. Application of SVD to these flattened maps spreads the signal across many of the 30 singular vectors, but a rotation of the vectors partitions the large majority of the signal into only five singular vectors. Fitting the time-dependent vectors to a sum of simple exponentials suggests that a chemical kinetic mechanism can describe the time-dependent structural data. Procedures for the effective SVD analysis of experimental time-resolved crystallographic data have been established and emphasize the necessity for minimizing systematic errors by modification of the data-collection protocol.

Cryophotolysis of ortho-nitrobenzyl derivatives of enzyme ligands for the potential kinetic crystallography of macromolecules.

Photolabile precursors of biologically active molecules (caged compounds) have been developed to allow temporally and spatially controlled release of bioactive compounds by rapid photolysis. [1] Caged compounds can be used to photochemically trigger an enzymatic reaction within an enzyme crystal and are thus valuable tools for studying protein dynamics at the atomic level by time-resolved X-ray crystallography. [2] Real-time-resolved X-ray crystallographic studies of fast biological processes have been applied mainly to a series of naturally light-sensitive proteins by using rapid data collection techniques (Laue diffraction) combined with high-intensity synchrotron X-ray beams. [3] However, caged ligands can also be used as photochemical triggers to capture transient structural species within enzyme crystals, provided photofragmentation reactions are fast, synchronous, and homogeneous throughout the crystals. The few successful examples which have been described so far concern enzymes whose reaction rates in the crystal were slow enough to allow both completion of the photolytic reaction and generally of the Laue data collection. [4] Alternatively, enzyme mutants may be produced to further increase the reaction intermediates’ lifetime. [5] Unfortunately, a series of technical pitfalls are associated with this method, including difficulties with the Laue method (essentially sensitivity to crystal disorder) [6]

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

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