J. W. Campbell

LAUEGEN version 6.0 and INTLDM

LAUEGEN version 6.0 is a significantly developed version of the original LAUEGEN program [Campbell (1995a). J. Appl. Cryst. 28, 228–236]. It is an X-Windows-based program which now carries out the stages of processing Laue diffraction data up to and including the integration of spot intensities. The integration routines have also been incorporated in a stand-alone integration program INTLDM. The programs are distributed as part of the Daresbury Laboratory Laue Software Suite.

LSCALE – the new normalization, scaling and absorption correction program in the Daresbury Laue software suite

A new program in the Daresbury Laue software 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.

Calcium binding sites in tomato bushy stunt virus visualized by laue crystallography

We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals.

The assembly mechanism of the lactate dehydrogenase tetramer from Bacillus stearothermophilus; the equilibrium relationships between quaternary structure and the binding of fructose 1,6-biphosphate, NADH and oxamate

Abstract (1) Unliganded lactate dehydrogenase from B. stearothermophilus exists in two states of quaternary structure, a dimer and a tetramer. The K d for the dimer-dimer interaction is 33 μM (expressed as a concentration of dimer). (2) The tetrameric state is stabilized by the tight binding of two fructose 1,6-bisphosphate molecules with a microscopic K d of 6–8 μM. (3) The state of subunit association has no influence on the binding of NADH ( K d = 1.5 μ M) but has a marked effect on the affinity of the active site for oxamate; a close structural analogue of pyruvate and competitive inhibitor of the enzyme. (4) In the tetrameric state stabilized with fructose 1,6-bisphosphate, the K d for oxamate is 0.013 mM, whereas the dimer binds the molecule only weakly ( K d = 2.2 mM ). Even in the absence of this effector, oxamate binds more tightly to the tetramer than to the dimer. This demonstrates that dimer-dimer association alone is sufficient to increase the affinity of the enzyme for oxamate. The K d for the association of oxamate with the unstabilized tetramer is less than 0.25 mM. (5) In the presence of fructose 1,6-bisphosphate the properties of the enzyme closely resemble those of the intensively studied porcine lactate dehydrogenases in the following respects; (a) the K d for NADH, (b) the K d for oxamate, (c) the ordered nature of coenzyme and substrate binding, (d) the quench in tryptophan fluorescence on binding NADH, (e) the quench in bound NADH fluorescence when the active site is occupied with oxamate.

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.

Structure of a phosphoglycerate mutase:3-phosphoglyceric acid complex at 1.7 A.

The crystal structure of the tetrameric glycolytic enzyme phosphoglycerate mutase from the yeast Saccharomyces cerevisiae has been determined to 1.7 A resolution in complex with the sugar substrate. The difference map indicates that 3-phosphoglycerate is bound at the base of a 12 A cleft, positioning C2 of the substrate within 3.5 A of the primary catalytic residue, histidine 8.

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

The parameters d min and λ min , 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.

The Laue Data Module (LDM) – a software development for Laue X-ray diffraction data processing

The Laue Data Module (LDM) has been defined and implemented in Fortran as the basis for new developments in the processing of Laue X-ray diffraction data. It provides a program-independent way of handling, storing and accessing the parameters required in the initial stages of Laue data processing. The program-independent Laue Reflection List (LRL) and associated functions have also been developed. The way in which these two developments may be used in application programs and as the basis for developing further program-independent Laue-data processing functions is described.

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 (2Fo−Fc) 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.

Application of energy-resolved measurements to Laue diffraction: determination of unit-cell parameters, deconvolution of harmonics and assignment of systematic absences.

The use of energy-resolved area detection of Laue diffraction patterns for the determination of unit-cell parameters and systematic absences is demonstrated. Seven different crystals having previously known unit cells were re-examined using Laue diffraction methods. These crystals included four different crystal systems including cubic, orthorhombic, tetragonal and monoclinic cells. The crystals had cell sizes ranging from 179.4 to 4588.3 A(3). Comparison of known and re-determined cells showed good agreement (ratio of known to measured cells = 0.987 +/- 0.18). A single procedure was suitable for all unit-cell determinations. The accuracy of the method is presently limited by the quality of the available energy measurements. Some of the crystals represent space groups containing systematic absences normally obscured by harmonic overlap when using the Laue method. These include absences due to 2(1) screw axes (h, k or 1 = 2n + 1) and cell centering (h + k = 2n + 1). All systematic absences were identified using a combination of multiple linear regression with either stepwise elimination or stepwise inclusion and an F test for assignment of systematic absence. The methods are discussed in detail and simulations are used to evaluate critical tolerances for future systems.