A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies - on ribosomes and on the allosteric protein aspartate transcarbamoylase - illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.

http://www.eng.uc.edu/~beaucag/Classes/Scattering/NativeStateSvergunSANSSAXS2003QRBP.pdf

Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution.

A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30–50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOW pipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.

/pdf/versatile-sample-environments-and-automation-for-biological-2blmf63qvj.pdf

Versatile sample environments and automation for biological solution X‐ray scattering experiments at the P12 beamline (PETRA III, DESY)

Small-angle X-ray scattering for macromolecules in solution is now widely used in structural biology to complement high-resolution structure determination obtained by X-ray crystallography or NMR. In the context of third-generation synchrotron sources, this increasing interest leads to developments in sample environments and automation. The presence of an online purification system is justified by the need for sample monodispersity. A combined system including an auto-sampler robot and online high-performance liquid chromatography (HPLC) has been developed and optimized at the SWING beamline of Synchrotron SOLEIL (Gif-sur-Yvette, France). In the sample changer mode, a few microlitres of sample can be injected between two air bubbles and circulated at a controlled speed of typically 40 µl min−1. A maximum of 14 samples per hour could be measured in this mode by remote controlling the sample injections. In the HPLC mode, an initially polydisperse sample can be separated into each of its components before immediate data acquisition. The sample cell is thermostated, and offers a visualization control and online UV–Vis absorption monitoring.

Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline

Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 644 A During protein export, one of the two oligosaccharides of NS1 is processed into an endo-beta-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell

Dengue Virus Type 1 Nonstructural Glycoprotein NS1 Is Secreted from Mammalian Cells as a Soluble Hexamer in a Glycosylation-Dependent Fashion

NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers

An evacuated, temperature-controlled cell has been built for use on the small-angle X-ray scattering instrument D24 at the synchrotron radiation facility LURE. The sample is placed in a quartz capillary sealed in a stainless-steel holder using a vacuum-tight glue. Several O rings provide a vacuum path upstream and downstream from the cell, so that the X-ray beam only meets the capillary walls and the solution under study between the slits and the beam stop, while the sample is maintained under atmospheric pressure. The cell temperature is controlled via a water circulation through a copper sheath in tight contact with the steel holder. The use of this cell results in a marked reduction of the background, as observed in two series of parallel experiments using a conventional cell and this evacuated cell. The decrease ranges from a factor of 2 at s 1 values larger than 0.008 A−1 to more than 15 at s = 0.00116 A−1, where s is the modulus of the scattering vector (s = 2sin θ/λ, 2θ is the scattering angle and λ is the wavelength of the X-rays).

J.-M. Dubuisson

Papers

Improved Signal‐to‐Background Ratio in Small‐Angle X‐ray Scattering Experiments with Synchrotron Radiation using an Evacuated Cell for Solutions