Laue Data Sets Research Articles

Direct observation of the excited state structure of a Ag(I)-Cu(I) complex

Heterodentate coordination complexes have been extensively studied because of their rich electronic and luminescent properties, which are of importance in the design of molecular devices. The short metal-metal contacts found in such complexes determine the nature of the lowest lying emissive states, and must be explored in order to understand their physical properties. Recent advances in time-resolved (TR) synchrotron techniques supported by specific data collection strategies and data processing procedures [1] allow for elucidation of molecular excited state geometries in the solid state. The approach has been so far successfully applied to several high-quality Laue-data sets collected at the 14-ID BioCars beamline at the Advanced Photon Source.[2] In this contribution we present synchrotron TR experiment results obtained for a new solvent-free crystal form of a model complex containing Ag(I) and Cu(I) (Ag2Cu2L4, L = 2-diphenylphosphino-3-methylindole).[3] This system exhibits red solid-state luminescence with a lifetime of about 1 µs. This is one of the shortest-lived excited states we have studied so far with the Laue technique. The relatively short lifetime goes along with significant structural changes observed upon irradiation, such as, the Ag...Ag distance shortening of about 0.2 Å in the excited state. The results clearly show strengthening of the Ag...Ag interactions suggesting a bond formation upon excitation. The photocrystallographic findings are supported by spectroscopic measurements and quantum computations. The results confirm the triplet nature of the emissive state originating mainly from a ligand-to-metal charge transfer. Research funded by the NSF (CHE1213223). BioCARS Sector 14 is supported by NIH, National Center for Research Resources (RR007707). APS is funded by the U.S. DOE, Office of Basic Energy Sciences (W-31-109-ENG-38). KNJ is supported by the Polish Ministry of Science and Higher Education through the "Mobility Plus" program.

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.

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.

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.

An Integration Routine Based on Profile Fitting with Optimized Fitting Area for the Evaluation of Weak and/or Overlapped Two-Dimensional Laue or Monochromatic Patterns

New software for the integration of weak and/or spatially overlapped diffraction patterns is presented. The program, named PROW (profile fitting for overlapped and weak data), uses box integration with an optimized summation area for strong nonoverlapped spots, and profile fitting with an optimized fitting area for weak and/or overlapped spots. The sizes of the integration areas are dynamically adjusted to find the best compromise between minimal bias resulting from statistical fluctuations of the X-ray background and maximal use of the information content in each diffraction spot. Deconvolution of spatially overlapped spots is performed by a standard least-squares procedure. The program is compatible with the Daresbury Laue software suite and with the DENZO package. It has been tested on several data-sets recorded on image-plate and CCD (charge-coupled device) detectors. Intensities extracted from very weak Laue data-sets containing as many as 60% overlapped spots were shown to be of sufficient quality to lead to an accurate structure refinement. Difficult monochromatic data-sets were also processed successfully, leading to a generally better compromise between reliability factors, redundancy and completeness than previously achieved. Concomitant improvement in atomic model refinement was also observed.

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.

Deconvolution of Energy Overlaps in Laue Diffraction

The energy-overlap problem in Laue diffraction often makes a Laue diffraction data set incomplete in a systematic way: low-resolution reflections more commonly overlap with other reflections than do reflections at higher resolution. We describe the development and testing of a harmonic deconvolution procedure that resolves energy overlaps accurately and is based on the wavelength-normalization curve obtained from single reflections. The conditions for satisfactory harmonic deconvolution are identified by examination of a series of data sets that differ in their redundancy. This procedure has been incorporated in the software system LaueView [Ren & Moffat (1995). J. Appl. Cryst. 28, 461–481]. Results on Laue data sets from crystals of lysozyme and α-haemolysin demonstrate that Laue data sets can be more than 90% complete even at 10 Å resolution and that structure amplitudes derived from these deconvoluted multiples can be as accurate as those derived from the best monochromatic data.

Quantitative Analysis of Synchrotron Laue Diffraction Patterns in Macromolecular Crystallography

The reduction of X-ray diffraction data obtained by the Laue method to accurate integrated intensities is more complicated and much less familiar than the reduction of monochromatic data. Problems of data accuracy and completeness have hindered the wide use of the Laue technique in macromolecular crystallography. Its unique advantage, data-collection speed, has been exploited only in situations such as fast time-resolved crystallography, to which monochromatic techniques are not as well suited. This paper reviews the major problems in data reduction in the Laue technique and provides a unified solution to the problems in integration of both streaky and spatially overlapping spots and data scaling. This solution has been incorporated into a new Laue diffraction data-reduction software package, LaueView. Laue data sets from crystals of lysozyme and α-haemolysin have been processed to test this solution, and demonstrate that Laue data sets can be reduced to yield structure amplitudes of at the very least the same quality as the best monochromatic data sets in terms of both accuracy and completeness.

Determination of dmin and λmin from the Intensity Distributions of Laue Patterns

The parameters dmin and λmin, essential for the processing of synchrotron X-ray Laue patterns, can be efficiently estimated by examination of the distribution of measured intensities as a function of d spacing and wavelength, respectively. A procedure that can be carried out automatically as one step in the data processing has been tested with Laue data sets from an organometallic compound and two proteins.

Laue Crystallography for Studying Rapid Reactions

Precise quantitation of static and time-resolved Laue diffraction patterns is undeniably more complex than for monochromatic patterns. Recent advances in integration and scaling algorithms demonstrate that, with suitable care in the conduct of the Laue experiment itself, Laue data sets can be obtained which rival the best monochromatic data sets in accuracy and completeness. These algorithms deal in an integrated fashion with the several main problems of Laue diffraction patterns: the elongated spots which arise from mosaic crystals, the spatial overlaps which occur in crowded diffraction patterns, the energy overlaps which arise from the mapping of a central line in reciprocal space onto a single spot in detector space, and wavelength normalization.

Electron density maps of lysozyme calculated using synchrotron laue data comprising singles and deconvoluted multiples

We have used lysozyme as a test case to illustrate the application of a new method of estimating the intensities of Laue multiples reflection data in protein crystallography. Hen egg-white lysozyme, an enzyme with a single polypeptide chain of 129 amino acids, crystallizes in space group P43212 with cell parametersa=b=79·1 Å,c=37·9 Å. Laue image plate data were collected using synchrotron radiation on station 9·5 at Daresbury with a total exposure time of 0·95 seconds. The data processed were separated into two data sets comprising the singles and the combined singles and deconvoluted multiples. The method of deconvolution was that of Campbell and Hao (1993), which utilizes the intensity variation of theλ-curve. Electron density maps (2F o−F c) based on the two data sets are then compared. This comparison shows that the deconvoluted multiples do indeed contribute usefully to the continuity of the maps due to the improved completeness of the data. A number of map sections along the polypeptide chain, based on the two Laue data sets, are shown for comparison. These include Arg 5, His 15, Phe 38, Asp 52, Tyr 53, Pro 70, Trp 108 and the four disulphide bridges.

Synchrotron X-ray crystallography techniques: time-resolved aspects of data collection

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. These properties are exploited routinely now to measure data at high resolution from proteins or from large unit cells (e.g. viruses), particularly using a monochromatized short wavelength beam. The time needed to measure protein crystal data-sets (in rotation geometry) can be quick (hours or minutes) compared with laboratory sources (weeks or days). Even so, more rapid data collection is of interest for timeresolved macromolecular crystallography. White beam : stationary crystal (Laue) geometry at SR sources offers shorter exposure times (seconds and less with film). Laue data-sets can be sensitive to subtle structural differences. Technical challenges still presented by SR Laue patterns include the energy overlap of low-resolution data and the spatial overlap of spots, both of which affect the completeness of data-sets. Some energy deconvolution is already possible by the use of multiple films. The spatial overlap problem can be alleviated by the use of three-dimensional detectors, such as a ‘toast-rack’ of plates. Monitoring of a crystalline process, via the Laue pattern, requires time slicing detector systems (e.g. based on CCDS) to be developed.