Petra Pernot

Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution

A description of the new ESRF BioSAXS beamline is given. The beamline presented is dedicated to small-angle X-ray scattering of macromolecules in solution operating with a high-throughput sample-changer robot and automated data analysis for quality control and feedback.

High-resolution three-dimensional imaging of flat objects by synchrotron-radiation computed laminography

Computed laminography with synchrotron radiation is developed and carried out for three-dimensional imaging of flat, laterally extended objects with high spatial resolution. Particular experimental conditions of a stationary synchrotron source have been taken into account by a scanning geometry different from that employed with movable conventional laboratory x-ray sources. Depending on the mechanical precision of the sample manipulation system, high spatial resolution down to the scale of 1μm can be attained nondestructively, even for objects of large lateral size. Furthermore, high beam intensity and the parallel-beam geometry enables easy use of monochromatic radiation for optimizing contrast and reducing imaging artifacts. Simulations and experiments on a test object demonstrate the feasibility of the method. Application to the inspection of solder joints in a flip-chip bonded device shows the potential for quality assurance of microsystem devices.

BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments

A robotic sample changer for solution X-ray scattering experiments optimized for speed and to use the minimum amount of material has been developed. This system is now in routine use at three high-brilliance European synchrotron sites, each capable of several hundred measurements per day.

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.

Online data analysis at the ESRF bioSAXS beamline, BM29

High-throughput small-angle X-ray scattering on proteins in solution (bioSAXS) at synchrotron sources is a commonly used technique in structural biology, which relies on highly automated data acquisition. Data reduction and primary analysis for bioSAXS experiments consist of a well defined series of individual tasks, the automation of which allows a first easy assessment of the quality of collected data and the adjustment of collection strategies if necessary. This article describes both the logic and the technical implementation of the automated processing pipeline for bioSAXS data at the ESRF BM29 beamline using the EDNA framework.

ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments

The ISPyB information-management system for crystallography has been adapted to include data from small-angle X-ray scattering of macromolecules in solution experiments.

Investigation of pore initiation in metal foams by synchrotron-radiation tomography

Synchrotron-radiation tomography was used to investigate early foaming stages of aluminium alloys. Monochromatic radiation, high spatial resolution down to the micrometer scale, partial beam coherence, and holographic reconstruction techniques permit the distinction between different foam constituents which are not visible by other volume imaging techniques. In combination with three-dimensional image analysis, the differences in the pore initiation processes in two different aluminium alloys are shown. We find that, in powder compacts made from prealloyed AA6061 alloy powder, pores appear predominantly around the blowing agent particles whereas, in compacts made from a powder blend of Al and Si, pores tend to initiate around Si particles.

Salt Effects in the Formation of Self-Assembled Lithocholate Helical Ribbons and Tubes

The formation of self-assembled nanotubes is usually accounted for by anisotropic elastic properties of membranelike precursors. We present experimental data as evidence of the role played by electrostatics in the formation of self-assembled tubes in alkaline aqueous suspensions of lithocholic acid (LCA). Striking salt effects are characterized by comparing the rheological, dynamical, and scattering properties of systems prepared either in stoichiometric neutralization conditions (SC) of LCA or in a large excess of sodium hydroxide (EOC, experimentally optimized conditions) and finally, in two steps: stoichiometric neutralization followed by an appropriate addition of NaCl (AISC). The SC liquid system is originally made up of loose helical ribbons (previous transmission electron microscopy data), and upon aging they exhibit both intra- and interordering processes. Initially, the helical ribbons are loose and progressively wind around a cylinder (R = 330 Å) with their edges exposed to the solvent. They can be temporarily organized in a centered rectangular two-dimensional lattice (pgg, a = 224 Å, b = 687 Å). Upon further aging, the ribbons wind into more compact helical ribbons (or tubes with helical grooves): their edges are less-exposed and their ordering vanishes. Upon addition of NaCl salt (as in the AISC systems), the specific screening of the intra-aggregate electrostatic repulsions induces the closure of the ribbons into tubes (R(ext) = 260 Å, R(int) = 245 Å as in the EOC systems). Simultaneously with the closure of the ribbons into plain tubes, a drastic enhancement of their interconnectivity through van der Waals attractions develops. Eventually, gels are obtained with networks having hexagonal bundles of tubes.

The Upgrade Programme for the Structural Biology beamlines at the European Synchrotron Radiation Facility – High throughput sample evaluation and automation

Automation and advances in technology are the key elements in addressing the steadily increasing complexity of Macromolecular Crystallography (MX) experiments. Much of this complexity is due to the inter-and intra-crystal heterogeneity in diffraction quality often observed for crystals of multi-component macromolecular assemblies or membrane proteins. Such heterogeneity makes high-throughput sample evaluation an important and necessary tool for increasing the chances of a successful structure determination. The introduction at the ESRF of automatic sample changers in 2005 dramatically increased the number of samples that were tested for diffraction quality. This first generation of automation, coupled with advances in software aimed at optimising data collection strategies in MX, resulted in a three-fold increase in the number of crystal structures elucidated per year using data collected at the ESRF. In addition, sample evaluation can be further complemented using small angle scattering experiments on the newly constructed bioSAXS facility on BM29 and the micro-spectroscopy facility (ID29S). The construction of a second generation of automated facilities on the MASSIF (Massively Automated Sample Screening Integrated Facility) beam lines will build on these advances and should provide a paradigm shift in how MX experiments are carried out which will benefit the entire Structural Biology community.

High-resolution three-dimensional imaging by synchrotron-radiation computed laminography

The methodical development and first instrumental implementation of computed laminography / tomosynthesis using synchrotron radiation are presented. The technique was developed for three-dimensional imaging of flat and laterally extended objects with high spatial resolution. This paper introduces the fundamental principle of the imaging process and discusses the methods particularities in comparison to computed tomography and computed laminography / digital tomosynthesis. Introducing a simple scanning geometry adapted to the particular experimental conditions of synchrotron imaging set-ups (such as the stationary source and a parallel beam) allows us to combine the advantages of laminography and those provided by synchrotron radiation, for instance monochromatic radiation in order to avoid beam hardening artefacts, high beam intensity for achieving high spatial resolution and fast scanning times or spatial coherence for exploiting phase contrast. The potential of the method for three-dimensional imaging of microelectronic devices is demonstrated by examples of flip-chip bonded and wire-bonded devices.