Search

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small‐angle X‐ray scattering (SAXS) beamline that specializes in high‐throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X‐ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X‐ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light‐responsive samples. [ABSTRACT FROM AUTHOR]

Through an expansive international effort that involved data collection on 12 small‐angle X‐ray scattering (SAXS) and four small‐angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent‐subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å−1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q‐range to <0.5 Å−1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering‐profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å−1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size‐exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low‐ and high‐concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set. [ABSTRACT FROM AUTHOR]

The design of a multipurpose sample cell holder for the high‐throughput (HT) beamline B21 is presented. The device is compatible with the robot bioSAXS sample changer currently installed on BM29, ESRF, and P12 Petra IV synchrotrons. This work presents an approach that uses 3D‐printing to make hardware alterations which can expand the versatility of HT beamlines at low cost. [ABSTRACT FROM AUTHOR]

Amino acid functionalized perylene bisimides (PBIs) form self‐assembled structures in solution, the nature of which depends on the local environment. Using a high‐throughput photocatalysis setup, five PBIs are studied for the hydrogen evolution reaction (HER) under a range of conditions (pH and hole scavenger concentration) across 350 experiments to explore the relationship between supramolecular structure and photocatalytic activity. Using small angle X‐ray scattering (SAXS), NMR spectroscopy and ultraviolet‐visible (UV‐vis) absorption spectroscopy, it is shown that photocatalytic activity is determined by the nature of the self‐assembled aggregate that is formed, demonstrating the potential of self‐assembly to tune activity. There is a clear correlation between the presence of charged flexible cylindrical aggregates and the occurrence of photocatalytic H2 production, with UV–vis spectroscopy indicating that the most active structure type has a distinctive form of π‐aggregation which is proposed to enable efficient charge separation across multiple PBI units. [ABSTRACT FROM AUTHOR]

B21 is a small‐angle X‐ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi‐layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in‐vacuum Eiger 4M (Dectris) detector. A high‐performance liquid chromatography system and a liquid‐handling robot make it possible to load solution samples into a temperature‐controlled in‐vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å−1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013. [ABSTRACT FROM AUTHOR]

MX2 is an in‐vacuum undulator‐based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X‐rays in the energy range 4.8–21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s−1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard‐to‐crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights. [ABSTRACT FROM AUTHOR]

Radiation damage is a major limitation to synchrotron small-angle X-ray scattering analysis of biomacromolecules. Flowing the sample during exposure helps to reduce the problem, but its effectiveness in the laminar-flow regime is limited by slow flow velocity at the walls of sample cells. To overcome this limitation, the coflow method was developed, where the sample flows through the centre of its cell surrounded by a flow of matched buffer. The method permits an order-of-magnitude increase of X-ray incident flux before sample damage, improves measurement statistics and maintains low sample concentration limits. The method also efficiently handles sample volumes of a few microlitres, can increase sample throughput, is intrinsically resistant to capillary fouling by sample and is suited to static samples and size-exclusion chromatography applications. The method unlocks further potential of third-generation synchrotron beamlines to facilitate new and challenging applications in solution scattering. [ABSTRACT FROM AUTHOR]

MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented. [ABSTRACT FROM AUTHOR]

Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B5) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein-protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface. [ABSTRACT FROM AUTHOR]

The SAXS/WAXS beamline at the Australian Synchrotron is an advanced and flexible undulator X-ray scattering beamline used for small- and wide-angle X-ray scattering analysis on a wide variety of solids, fluids and surfaces across a diverse range of research and development fields. The beamline has numerous features that minimize the intensity of the instrument background, provide automated stable optics, and allow accurate analysis of very weakly scattering samples. The geometric and intensity requirements of a three-slit collimation system are described in detail for conventional metal and single-crystal germanium slits. Straightforward ray tracing and simple linear projections describe the observed direct beam as well as parasitic background scattering geometry of the beamline at its longest camera length, providing a methodology for the design and operation of similar beamlines. As an aid to instrument design, the limit of background intensity determined by the intensity incident on single-crystal germanium guard slit edges and its q dependence was quantified at 11 keV. Details of the beamline's implementation, underlying optical concept and measured performance are given. [ABSTRACT FROM AUTHOR]

The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the 'Toblerone' fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser- Lys catalytic dyads. A single catalytic dyad ( Ser85- Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure-function relationship. [ABSTRACT FROM AUTHOR]

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL ( SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. [ABSTRACT FROM AUTHOR]

Following the discovery of an exo-1,3/1,4-β-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue β- d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. Database Structural data are available in the Protein Data Bank under the accession numbers , , and [ABSTRACT FROM AUTHOR]

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]