Biological Small-angle X-ray Scattering Research Articles

Inline small-angle X-ray scattering-coupled chromatography under extreme hydrostatic pressure.

As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small-angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography-coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. The apparatus described here functions at a range of temperatures (0°C-50°C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

Rational Designed Hybrid Peptides Show up to a 6-Fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides Measured by BioSAXS

Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a “dirty drug” model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

BioSAXS on the SWING beamline at Synchrotron SOLEIL

Small-angle X-ray scattering (SAXS) of proteins in solution has become a key tool for biochemists and structural biologists, thanks especially to the availability of beamlines with high-throughput capabilities at synchrotron sources. Despite the large spectrum of scientific disciplines tackled on the SWING beamline since its opening in 2008, there has always been a strong commitment to offering state-of-the-art biological SAXS (BioSAXS) instrumentation and data reduction methods to the scientific community. The extremely reliable in-vacuum EigerX-4M detector allows collection of an unlimited number of frames without noise. A small beamstop including a diamond diode-based monitor enables measurements of the transmitted intensity with 0.1% precision as well as a q max/q min ratio as large as 140 at a single distance. The parasitic scattering has been strongly reduced by the installation of new hybrid blades. A new thermally controlled in-vacuum capillary holder including fibre-optics-based spectroscopic functionalities allows the simultaneous use of three spectroscopic techniques in addition to SAXS measurements. The addition of a second high-performance liquid chromatography (HPLC) circuit has virtually eliminated the waiting time associated with column equilibration. The easy in-line connection of a multi-angle light scattering spectrometer and a refractometer allows for an independent determination of the molecular mass and of the concentration of low-UV-absorption samples such as detergents and sugars, respectively. These instrumental improvements are combined with important software developments. The HPLC injection Agilent software is controlled by the SAXS beamline acquisition software, allowing a virtually unlimited series of automated SAXS measurements to be synchronized with the sample injections. All data-containing files and reports are automatically stored in the same folders, with names related to both the user and sample. In addition, all raw SAXS images are processed automatically on the fly, and the analysed data are stored in the ISPyB database and made accessible via a web page.

Improving data quality and expanding BioSAXS experiments to low-molecular-weight and low-concentration protein samples.

The addition of compounds to scavenge the radical species produced during biological small-angle X-ray scattering (BioSAXS) experiments is a common strategy to reduce the effects of radiation damage and produce better quality data. As almost half of the experiments leading to structures deposited in the SASBDB database used scavengers, finding potent scavengers would be advantageous for many experiments. Here, four compounds, three nucleosides and one nitrogenous base, are presented which can act as very effective radical-scavenging additives and increase the critical dose by up to 20 times without altering the stability or reducing the contrast of the tested protein solutions. The efficacy of these scavengers is higher than those commonly used in the field to date, as verified for lysozyme solutions at various concentrations from 7.0 to 0.5 mg ml-1. The compounds are also very efficient at mitigating radiation damage to four proteins with molecular weights ranging from 7 to 240 kDa and pH values from 3 to 8, with the extreme case being catalase at 6.7 mg ml-1, with a scavenging factor exceeding 100. These scavengers can therefore be instrumental in expanding BioSAXS to low-molecular-weight and low-concentration protein samples that were previously inaccessible owing to poor data quality. It is also demonstrated that an increase in the critical dose in standard BioSAXS experiments leads to an increment in the retrieved information, in particular at higher angles, and thus to higher resolution of the model.

Recent Developments at the Beamline for Biological Small Angle x-ray Scattering BL4-2 at SSRL

Recent Developments at the Beamline for Biological Small Angle x-ray Scattering BL4-2 at SSRL

Biological Small-Angle X-ray Scattering Combined with Size-exclusion Chromatography

Biological Small-Angle X-ray Scattering Combined with Size-exclusion Chromatography

Upgraded SSRF BL19U2 beamline for small-angle X-ray scattering of biological macromolecules in solution

The biological small-angle X-ray scattering (BioSAXS) beamline (BL19U2) at the Shanghai Synchrotron Radiation Facility, China, is dedicated exclusively to small-angle scattering experiments for biological macromolecules in solution. With recent advances in data-analysis algorithms and X-ray detectors, SAXS becomes an ideal complementary technique to other structural and biophysical methods, but it can also be applied alone to obtain important structural information. Owing to the increasing interest in solution scattering studies from the biological community, the workload on BL19U2 has steadily risen. A major upgrade of BL19U2 was performed to improve the beamline data quality, to enrich the possible sample environments and to provide a user-friendly interface. These upgrades involved the major components of BL19U2, including the optical system (slits, beamstop), the electronics, the control and acquisition software, and the sample environments, which resulted in improvements to the collected angular range in BL19U2. These upgrades have significantly broadened the scope of macromolecule size (from kilodaltons to gigadaltons) analysed at the beamline. The dedicated BL19U2 BioSAXS beamline now offers fully automated data-collection and remote-control possibilities. These developments have paved the way for high-throughput studies that generate significant quantities of structure information over a short period of time.

Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions.

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters di (di = 1.7 mm, close to optimal for 10 keV; and di = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.

In Situ Structural Changes of Biological Macromolecules with BioSAXS

Biological small angle X-ray scattering (BioSAXS) is a nowadays well-established technique to study low resolution structural changes within biological macromolecules in their native environment. In addition, SAXS is a powerful tool for structural characterization and the quantitative analysis of flexible system as a highly complementary method to the high resolution methods of X-ray crystallography and NMR. In general, BioSAXS allows studying three dimensional low resolution structures in different chemical environments under defined conditions (e.g., temperature, pH, dielectric properties, etc.) using not only ab initio methods but also rigid body modeling. Furthermore, the shape (form factor) and interaction potential (structure factor) of the active quaternary protein structure can be assessed using the pair distance distribution function (PDDF) with the use of the generalized indirect Fourier transformation (GIFT). Especially in situ measurements of biological macromolecules have gained a lot of attention in the last decades due to the related structural changes and correlated bioactivity (i.e., native and denatured state).As model substance, Lysozyme was analyzed in terms of its quaternary structural changes upon in situ heat treatment and changes in the dielectric constant of the chemical environment. The validation of the form and structure factor components of the analyzed system allows determining the influence of the changed environmental conditions on the protein structure. The changed chemical environment causes an elongation of the previously globular protein structure. In aqueous environment, Lysozyme is stabilized via electrostatic stabilization. In ethanolic solution, the stabilization mechanism is changed, resulting in a change of structure (e.g., elongation).

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline.

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

Online ion-exchange chromatography for small-angle X-ray scattering.

Biological small-angle X-ray scattering (BioSAXS) is a powerful technique to determine the solution structure, particle size, shape and surface-to-volume ratio of macromolecules. However, a drawback is that the sample needs to be monodisperse. To ensure this, size-exclusion chromatography (SEC) has been implemented on many BioSAXS beamlines. Here, the integration of ion-exchange chromatography (IEC) using both continuous linear and step gradients on a beamline is described. Background subtraction for continuous gradients by shifting a reference measurement and two different approaches for step gradients, which are based on interpolating between two background measurements, are discussed. The results presented here serve as a proof of principle for online IEC and subsequent data treatment.

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

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.

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell.

With the rise in popularity of biological small-angle X-ray scattering (BioSAXS) measurements, synchrotron beamlines are confronted with an ever-increasing number of samples from a wide range of solution conditions. To meet these demands, an increasing number of beamlines worldwide have begun to provide automated liquid-handling systems for sample loading. This article presents an automated sample-loading system for BioSAXS beamlines, which combines single-channel disposable-tip pipetting with a vacuum-enclosed temperature-controlled capillary flow cell. The design incorporates an easily changeable capillary to reduce the incidence of X-ray window fouling and cross contamination. Both the robot-control and the data-processing systems are written in Python. The data-processing code, RAW, has been enhanced with several new features to form a user-friendly BioSAXS pipeline for the robot. The flow cell also supports efficient manual loading and sample recovery. An effective rinse protocol for the sample cell is developed and tested. Fluid dynamics within the sample capillary reveals a vortex ring pattern of circulation that redistributes radiation-damaged material. Radiation damage is most severe in the boundary layer near the capillary surface. At typical flow speeds, capillaries below 2 mm in diameter are beginning to enter the Stokes (creeping flow) regime in which mixing due to oscillation is limited. Analysis within this regime shows that single-pass exposure and multiple-pass exposure of a sample plug are functionally the same with regard to exposed volume when plug motion reversal is slow. The robot was tested on three different beamlines at the Cornell High-Energy Synchrotron Source, with a variety of detectors and beam characteristics, and it has been used successfully in several published studies as well as in two introductory short courses on basic BioSAXS methods.

Software for the high-throughput collection of SAXS data using an enhancedBlu-Ice/DCScontrol system

Biological small-angle X-ray scattering (SAXS) provides powerful complementary data for macromolecular crystallography (MX) by defining shape, conformation and assembly in solution. Although SAXS is in principle the highest throughput technique for structural biology, data collection is limited in practice by current data collection software. Here the adaption of beamline control software, historically developed for MX beamlines, for the efficient operation and high-throughput data collection at synchrotron SAXS beamlines is reported. The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the biological SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide a potential route for other SAXS beamlines in need of robust and efficient beamline control software. As a great deal of effort and optimization has gone into crystallographic software, the adaption and extension of crystallographic software may prove to be a general strategy to provide advanced SAXS software for the synchrotron community. In this way effort can be put into optimizing features for SAXS rather than reproducing those that have already been successfully implemented for the crystallographic community.