Small-angle X-ray Scattering Methods Research Articles

Microfluidic Controlled Self-Assembly of Polylactide (PLA)-Based Linear and Graft Copolymers into Nanoparticles with Diverse Morphologies.

This study outlines the microfluidic (MF) controlled self-assembly of polylactide (PLA)-based linear and graft copolymers. The PLA-based copolymers (PLA-Cs) were synthesized through a convenient one-pot/one-step ROP/RAFT technique. Three distinct vinyl monomers-triethylene glycol methacrylate (TEGMA), 2-hydroxypropyl methacrylate (HPMA), and N-(2-hydroxypropyl) methacrylamide (HPMAA) were employed to prepare various copolymers: linear thermoresponsive polylactide-b-poly(triethylene glycol methacrylate) (PLA-b-PTEGMA), graft pseudothermoresponsive poly[N-(2-hydroxypropyl)] methacrylate-g-polylactide (PHPMA-g-PLA), and graft amphiphilic poly[N-(2-hydroxypropyl)] methacrylamide-g-polylactide (PHPMAA-g-PLA). The MF technology was utilized for the controlled self-assembly of these PLA-based BCs in a solution, resulting in a range of nanoparticle (NP) morphologies. The thermoresponsive PLA-b-PTEGMA diblock copolymer formed thermodynamically stable micelles (Ms) through kinetically controlled assemblies. Similarly, employing MF channels led to the self-assembly of PHPMA-g-PLA, yielding polymersomes (PSs) with adjustable sizes under the same solution conditions. Conversely, the PHPMAA-g-PLA copolymer generated worm-like particles (Ws). The analysis of resulting nano-objects involves techniques such as transmission electron microscopy, dynamic light scattering investigations (DLS), and small-angle X-ray scattering (SAXS). More specifically, the thermoresponsive behavior of PLA-b-PTEGMA and PHPMA-g-PLA nano-objects is validated through variable-temperature DLS, TEM, and SAXS methods. Furthermore, the study explored the specific interactions between the formed Ms, PSs, and/or Ws with proteins in human blood plasma, utilizing isothermal titration calorimetry.

Microstructural analysis of organic matter in shale by SAXS and WAXS methods

Characterizing the kerogen-hosted pore structures is essential to understand the adsorption, transport and storage potential in organic-rich shale reservoirs. In this paper, we first separated the organic matter (kerogen) from the mineral matrix in four different shale samples of the Bakken Formation with different thermal maturities and then analyzed their chemical compositions using the wide-angle X-ray scattering (WAXS) method. Next, we acquired small-angle X-ray scattering (SAXS) to characterize the structure of the organic matter and see how these two will relate. The WAXS results showed that the isolated kerogens have high purity (free of inorganic minerals) and retain different chemical compositions. Moreover, SAXS analysis revealed that the isolated kerogens have similar radius of gyration (Rg) which is around 90 Å and the molecules are in the compact mode. Based on the pore size distribution analysis from the SAXS data, two main peaks were found in all of these four samples with one peak less than 40 Å and the other one larger than 1000 Å. Also, the TEM images revealed that Sample 1 is abundant in pores with sizes around 20 nm while Sample 2 does not have pores of that size, which agrees with the results from the pore size distribution that was obtained from the SAXS method. Ultimately, this study exhibits how different analytical instruments can provide us with useful information from complex structures of geomaterials.

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.

Pore morphology and topology of zeolite imidazolate framework ZIF‐67 revealed by small‐angle X‐ray scattering

AbstractThe pore characterization of ZIF system using small‐angle X‐ray scattering (SAXS) technique is presented for the first time. The pore characterization of ZIF‐67 framework structure as model system using the conventional nitrogen adsorption and SAXS methods was performed in this work. The comparison between the results obtained by two methods can provide the verification, complementary information, and error analysis to the conventional method using nitrogen atom as probe. The pore network and morphology are subjected to the topology of ZIF framework structure. The theoretical calculation of pore morphology in the unit cage of ZIF‐67 structure is verified by this SAXS characterization. This SAXS study can be used as the reference of modeling pore for general ZIF systems.

SAXS methods for investigating macromolecular and self-assembled polyelectrolyte complexes.

Polyelectrolyte complexation is driven by associative interactions between oppositely charged polyelectrolytes, resulting in formation of a macroscopic polymer dense phase and a polymer dilute phase with applications in coatings, adhesives, and purification membranes. Beyond macroscale phase separation, precision polymer synthesis has enabled further development of polyelectrolyte complex (PEC)-based self-assembled micelles and hydrogels with applications in biotechnology. Interestingly, it has been suggested that mechanisms similar to polyelectrolyte complexation drive formation of biological condensates that play an indispensable role in cellular biogenesis. The formation pathways and functionality of these complex materials is dependent on the physical properties that are built into polymer structure and the resulting physical conformation in the dilute and dense phase. Scattering techniques have enabled in situ investigation of structure-function relationships in PEC materials that may address unresolved biophysical questions in cellular processes as well as catalyze the development of novel materials for diverse applications. We describe preparation of PEC materials with controlled polymer characteristics (length, blockiness, charge density), small-angle X-ray scattering (SAXS) techniques employed to probe appropriate length scales, and the data analysis routines from a practical standpoint for new users. This article deals with bulk complexes and not with the related, important and interesting area of non-equilibrium layer-by-layer assembly of polyelectrolytes.

Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29–121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical ‘tip motif’. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P2-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P2 analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P2 and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P2-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1–287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.

Structural analysis of polymer thin films using GISAXS in the tender X-ray region: Concept and design of GISAXS experiments using the tender X-ray energy at BL-15A2 at the Photon Factory

If small angle X-ray scattering (SAXS) utilizing the soft X-ray region is available, advanced and unique experiments, which differ from traditional SAXS methods, can be realized. For example, grazing-incidence small angle X-ray scattering (GISAXS) using hard X-ray is a powerful tool for understanding the nanostructure in both vertical and lateral directions of thin films, while GISAXS utilizing the tender X-ray region (SX-GISAXS) enables depth-resolved analysis as well as a standard GISAXS analysis in thin films. Thus, at BL-15A2 at the Photon Factory, a dedicated diffractometer for SX-GISAXS (above 2.1 keV) was constructed. This diffractometer is composed of four vacuum chambers and can be converted into the vacuum state from the sample chamber in front of the detector surface. Diffractions are clearly observed until 12th peak when measuring collagen by SAXS with an X-ray energy of 2.40 keV and a camera length of 825 mm. Additionally, we conducted the model experiment using SX-GISAXS with an X-ray energy of 2.40 keV to confirm that a poly(methyl methacrylate)-poly(n-butyl acrylate) block copolymer thin film has a microphase-separated structure in the thin film, which is composed of lamellae aligned both parallel and perpendicular to the substrate surface. Similarly, in a polystyrene-poly(methyl methacrylate) block copolymer thin film, SX-GISAXS with 3.60 keV and 5.73 keV revealed that hexagonally packed cylinders are aligned parallel to the substrate surface. The incident angle dependence of the first order peak position of the qz direction obtained from experiments at various incident X-ray energies agrees very well with the theoretical one calculated from the distorted wave Born approximation.

Facilitating model reconstruction for single-particle scattering using small-angle X-ray scattering methods.

X-ray free-electron lasers generate intense femtosecond X-ray pulses, so that high-resolution structure determination becomes feasible from noncrystalline samples, such as single particles or single molecules. At the moment, the orientation of sample particles cannot be precisely controlled, and consequently the unknown orientation needs to be recovered using computational algorithms. This delays the model reconstruction until all the scattering patterns have been re-oriented, which often entails a long elapse of time and until the completion of the experiment. The scattering patterns from single particles or multiple particles can be summed to form a virtual powder diffraction pattern, and the low-resolution region, corresponding to the small-angle X-ray scattering (SAXS) regime, can be analysed using existing SAXS methods. This work presents a pipeline that converts single-particle data sets into SAXS data, from which real-time model reconstruction is achieved using the model retrieval approach implemented in the software package SASTBX [Liu, Hexemer & Zwart (2012). J. Appl. Cryst.45, 587-593]. To illustrate the applications, two case studies are presented with real experimental data sets collected at the Linac Coherent Light Source.

X-ray scattering methods for accurate analyses of flexible complexes

A grand challenge for structural biology is to efficiently inform macromolecular functions that involve flexible or unstructured regions and require multiple conformational states including membrane proteins and protein-nucleic acid complexes. Small angle X-ray scattering (SAXS) can probe at resolutions sufficient to distinguish conformational states, characterize flexible macromolecules, and screen in high-throughput under most solution conditions. However, methods for analyzing SAXS data and models have restricted progress. We are therefore developing SAXS methods that provide high-throughput, quantitative and superposition-independent evaluation of solution-state conformations and quantitatively define flexibility and disorder. The SIBYLS beamline provides hardware and software to integrate SAXS and crystal structure results. Our SAXS methods aim to improve crystallization and interpretation of crystal structures and NMR structure quality. We have invented a statistically robust method for assessing model-data agreements (chi-square free) akin to cross-validation. We also developed a metric and method for rapid quantitative and comprehensive assessment of molecular similarity suitable to examine functionally important conformational changes. To extend SAXS analysis to low concentrations and complex mixtures, we are developing SAXS with gold nano-crystal labels to enable examination of protein-induced DNA distortions along pathways key to the DNA repair, replication, transcription, and packaging. Collective results suggest SAXS can provide accurate shapes, assembly states, and comprehensive conformations of flexible complexes in solution that inform biology in fundamental ways.

Small-angle X-ray scattering to obtain models of multivalent lectin-glycan complexes.

Recent advances in small-angle X-ray scattering (SAXS) have led to the ability to model the glycans on glycoproteins and to obtain the low-resolution solution structures of complexes of lectins bound to multivalent glycan-presenting scaffolds. This progress in SAXS can respond to the increasing interest in the biological action of glycoproteins and lectins and in the design of multivalent glycan-based antagonists. Carbohydrates make up a significant part of the X-ray scattering content in SAXS and should be included in the model together with the protein, whose structure is most often based on a crystal structure or NMR ensemble, to give a far-improved fit with the experimental data. The modeling of the spatial positioning of glycans on proteins or in the architecture of lectin-glycan complexes delivers low-resolution structural information hitherto unmatched by any other method. SAXS data on the bacterial lectin FimH, strongly bound to heptyl α-D-mannose on a sevenfold derivatized β-cyclodextrin, permitted determination of the stoichiometry of the complex and the geometry of the lectin deposition on the multivalent β-cyclodextrin. The SAXS methods can be applied to larger complexes as the technique imposes no limit on the size of the macromolecular assembly in solution.

Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source.

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world's mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B(4)C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.

Aggregation behavior of pyridinium based ionic liquids in water – Surface tension, 1H NMR chemical shifts, SANS and SAXS measurements

The aggregation behavior of short alkyl chain ionic liquids (ILs), namely 1-butyl, or 1-hexyl or 1-octylpyridinium and 1-octyl-2-, or -3-, or -4-methylpyridinium chlorides, in water has been assessed using surface tension, electrical conductance, 1H NMR, small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) measurements. Critical aggregation concentrations (CACs), adsorption (at air/water interface) and thermodynamic parameters of aggregation have been reported. The values of CAC and area per adsorbed molecule decrease with the number of carbon atoms in the alkyl chain. The aggregation process is driven by both favorable enthalpy and entropy contributions. An attempt was made to examine the morphological features of the aggregates in water using SANS and SAXS methods. SANS and SAXS curves displayed diffuse structural peaks that could not be model fitted, and therefore, we calculated the mean aggregation numbers from the Qmax assuming that IL molecules typically order into cubic type clusters.

X-ray scattering from unilamellar lipid vesicles

An improved small-angle X-ray scattering (SAXS) method for determining asymmetric lipid bilayer structure in unilamellar vesicles is presented. From scattering theory, analytic expressions are derived for the bilayer form factor over flat and spherical geometries, assuming the lipid bilayer electron density to be composed of a series of Gaussian shells. This is in contrast to both classic diffraction and Guinier hard-shell SAXS methods which, respectively, are capable only of ascertaining symmetric bilayer structure and limited-resolution asymmetric structure. Using model fitting and direct calculation of the form factor, using only one equation, an asymmetric electron density profile of the lipid vesicle is obtained with high accuracy, as well as the average radius. The analysis suggests that the inner leaflet of a unilamellar lipid vesicle is `rougher' than the outer one.

Characterization of the Microdomain Structure in Polystyrene−Polyisoprene Block Copolymers by 1H Spin Diffusion and Small-Angle X-ray Scattering Methods

1H spin diffusion and small-angle X-ray scattering (SAXS) measurements were carried out for a series of polystyrene−polyisoprene block copolymers of varying molecular weight and molecular architecture. The sizes of domains and the thickness of the interphase in the copolymers were determined from the spin diffusion measurements using a model which explicitly considers the effects of 1H spin−lattice relaxation. The thickness of the interphase determined by NMR was found to be constant (ca. 3 nm) for all of the copolymers. The variations in domain size and intersphere distance as a function of molecular weight were found to be in good agreement with those determined by the more established SAXS methods. However, the intersphere distances measured by NMR were found to be consistently smaller than those measured by SAXS, and this difference may be caused by simplifications in the morphological model used in the analysis of the spin diffusion curves.

Surface fractal dimension of microporous carbon fibres by nitrogen adsorption

The surface fractal dimension of three kinds of microporous carbon fibre has been determined from adsorption data of organic molecules of different molecular sizes (MAI method), by detailed N2 adsorption isotherms with the aid of the recently proposed Avnir–Jaroniec theory (AJ method) and by small-angle X-ray scattering (SAXS). The application conditions of the AJ theory to the N2 adsorption isotherm have been examined. The surface fractal dimension by the AJ method depends on the range of the fractional filling. In the fractional filling range ca. 0.8–1.0 the value of the surface fractal dimension is ca. 2.6–2.7 and nearly constant; therefore, this range of adsorption data is suggested for evaluating the fractal dimension by the AJ method. The surface fractal dimension by the MAI method was 2.3–3.0 and that by SAXS was 2.6–2.8 in dry air. Thus the AJ method, which uses a single adsorption isotherm, can give a reasonable value of the surface fractal dimension, similar to those given by MAI and SAXS methods.

On the Size Distribution of Guinier‐Preston Zones Grown in an Al‐4.5 at.% Zn Alloy at Room Temperature

AbstractThe size distribution SD of the Guinier‐Preston (GP) zones grown in an Al‐4.5 at.% Zn alloy were determined by analysis of the intensities of the small‐angle X‐ray scattering (SAXS) curves. Four different methods frequently used in the literature were applied and the obtained results were compared. In addition the SD's obtained by the SAXS methods were compared with that found by high‐resolution electron microscopic investigations. – From these comparisons conclusions were drawn.