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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.

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]

Pluronic F127 dimethacrylate (F127-DM) inks exhibit potential for 3D printing biomaterials due to their combined gelation and photo-crosslinking capabilities. Previous studies have explored their application in extrusion 3D printing of medical devices, relying on their long-range micellar ordering to impart mechanical stability, stretchability, and control over drug delivery. However, there is a lack of information regarding the impact of the extrusion and photo-crosslinking processes on the micellar ordering of F127-DM. Herein, we employed in operando 3D printing synchrotron small-angle X-ray scattering (SAXS) to analyze the influence of various printing parameters, including nozzle shape, nozzle size, extrusion rate, ink composition, and photo-crosslinking, on the micellar ordering of F127-DM. Our findings from 2D SAXS patterns indicate orientation of the micellar packing clusters along the flow direction. The nozzle diameter and shape emerged as crucial parameters, leading to increased disruption of long-range ordering. Furthermore, 1D SAXS curves during UV-Vis light exposure and photo-crosslinking revealed a partial preservation of the micellar packing structure, with cluster size reduction, and coexisting with randomly distributed micelles and unimers. These results underscore the importance of in operando synchrotron experiments for the systematic study of 3D printing parameters from a nanostructure perspective. [ABSTRACT FROM AUTHOR]

Pluronic F127 dimethacrylate (F127-DM) inks exhibit potential for 3D printing biomaterials due to their combined gelation and photo-crosslinking capabilities. Previous studies have explored their application in extrusion 3D printing of medical devices, relying on their long-range micellar ordering to impart mechanical stability, stretchability, and control over drug delivery. However, there is a lack of information regarding the impact of the extrusion and photo-crosslinking processes on the micellar ordering of F127-DM. Herein, we employed in operando 3D printing synchrotron small-angle X-ray scattering (SAXS) to analyze the influence of various printing parameters, including nozzle shape, nozzle size, extrusion rate, ink composition, and photo-crosslinking, on the micellar ordering of F127-DM. Our findings from 2D SAXS patterns indicate orientation of the micellar packing clusters along the flow direction. The nozzle diameter and shape emerged as crucial parameters, leading to increased disruption of long-range ordering. Furthermore, 1D SAXS curves during UV-Vis light exposure and photo-crosslinking revealed a partial preservation of the micellar packing structure, with cluster size reduction, and coexisting with randomly distributed micelles and unimers. These results underscore the importance of in operando synchrotron experiments for the systematic study of 3D printing parameters from a nanostructure perspective.

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV–vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.

The function of biomolecules is tightly linked to their structure, and changes therein. Time-resolved X-ray solution scattering has proven a powerful technique for interrogating structural changes and signal transduction in photoreceptor proteins. However, these only represent a small fraction of the biological macromolecules of interest. More recently, laser-induced temperature jumps have been introduced as a more general means of initiating structural changes in biomolecules. Here we present the development of a setup for millisecond time-resolved X-ray solution scattering experiments at the CoSAXS beamline, primarily using infrared laser light to trigger a temperature increase, and structural changes. We present results that highlight the characteristics of this setup along with data showing structural changes in lysozyme caused by a temperature jump. Further developments and applications of the setup are also discussed.

Mixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori. To address this issue, the REGALS method (regularized alternating least squares) is introduced, which incorporates simple expectations about the data as prior knowledge, and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, making it well suited for exploring datasets with unknown species. Here, REGALS is applied to the analysis of experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing and time-resolved temperature jump. Based on its performance with these challenging datasets, it is anticipated that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and Python and is available freely as an open-source software package.

The function of biomolecules is tightly linked to their structure, and changes therein. Time‐resolved X‐ray solution scattering has proven a powerful technique for interrogating structural changes and signal transduction in photoreceptor proteins. However, these only represent a small fraction of the biological macromolecules of interest. More recently, laser‐induced temperature jumps have been introduced as a more general means of initiating structural changes in biomolecules. Here we present the development of a setup for millisecond time‐resolved X‐ray solution scattering experiments at the CoSAXS beamline, primarily using infrared laser light to trigger a temperature increase, and structural changes. We present results that highlight the characteristics of this setup along with data showing structural changes in lysozyme caused by a temperature jump. Further developments and applications of the setup are also discussed. [ABSTRACT FROM AUTHOR]

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 Å. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.

Viruses have been of interest to mankind since their discovery as small infectious agents in the nineteenth century. Because many viruses cause diseases to humans and agriculture, they were rigorously studied for biological and medical purposes. Viruses have remarkable properties such as the symmetry and self-assembly of their protein envelope, maturation into infectious virions, structural stability, and disassembly. Solution X-ray scattering can probe structures and reactions in solutions, down to subnanometer spatial resolution and millisecond temporal resolution. It probes the bulk solution and reveals the average shape and average mass of particles in solution and can be used to study kinetics and thermodynamics of viruses at different stages of their life cycle. Here we review recent work that demonstrates the capabilities of solution X-ray scattering to study in vitro the viral life cycle. [ABSTRACT FROM AUTHOR]