Small-angle X-ray Scattering Data Research Articles

Evaluating Anti-CD32b F(ab) Conformation Using Molecular Dynamics and Small-Angle X-Ray Scattering

Complementary strategies of small-angle x-ray scattering (SAXS) and crystallographic analysis are often used to determine atomistic three-dimensional models of macromolecules and their variability in solution. This combination of techniques is particularly valuable when applied to macromolecular complexes to detect changes within the individual binding partners. Here, we determine the x-ray crystallographic structure of a F(ab) fragment in complex with CD32b, the only inhibitory Fc-γ receptor in humans, and compare the structure of the F(ab) from the crystal complex to SAXS data for the F(ab) alone in solution. We investigate changes in F(ab) structure by predicting theoretical scattering profiles for atomistic structures extracted from molecular dynamics (MD) simulations of the F(ab) and assessing the agreement of these structures to our experimental SAXS data. Through principal component analysis, we are able to extract principal motions observed during the MD trajectory and evaluate the influence of these motions on the agreement of structures to the F(ab) SAXS data. Changes in the F(ab) elbow angle were found to be important to reach agreement with the experimental data; however, further discrepancies were apparent between our F(ab) structure from the crystal complex and SAXS data. By analyzing multiple MD structures observed in similar regions of the principal component analysis, we were able to pinpoint these discrepancies to a specific loop region in the F(ab) heavy chain. This method, therefore, not only allows determination of global changes but also allows identification of localized motions important for determining the agreement between atomistic structures and SAXS data. In this particular case, the findings allowed us to discount the hypothesis that structural changes were induced upon complex formation, a significant find informing the drug development process. The methodology described here is generally applicable to deconvolute global and local changes of macromolecular structures and is well suited to other systems.

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.

Direct shape determination of intermediates in evolving macromolecular solutions from small-angle scattering data.

Many important biological processes like amyloid formation, viral assembly etc. can be monitored in vitro. Small-angle X-ray scattering (SAXS) is one of the most effective techniques to structurally characterize these processes in solution. For monodisperse systems and some oligomeric mixtures, low-resolution shapes can be determined ab initio from the SAXS data, but for evolving systems, such analysis is hampered by the presence of multiple species and no direct reconstruction procedures are available. The authors consider a frequently occurring case where the scattering from the initial and final states of the process are known but there exists a major (unknown) intermediate component. A method is presented to directly reconstruct the low-resolution shape of this transient component together with its volume fractions from multiple scattering patterns recorded from an evolving system. The method is implemented in the computer program DAMMIX freely available to academic users and its effectiveness is illustrated in several synthetic and experimental examples.

Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering.

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.

Multilevel Morphology of Complex Nanoporous Materials.

This work exploits gas adsorption and small-angle X-ray scattering (SAXS) to determine the morphology of complex nanoporous materials. We resolve multiple classes of porosity including previously undetected large-scale texture that significantly compromises the canonical interpretation of gas adsorption. Specifically, a UVM-7 class mesoporous silica was synthesized that has morphological features on three length scales: macropores due to packing of 150 nm globules, 1.9 nm radius spherical mesopores inside the globules, and >7 nm pockets on and between the globules. The total and external surface areas, as well as the mesopore volume, were determined using gas adsorption (αs-plot) and SAXS. A new approach was applied to the SAXS data using multilevel fitting to determine the surface areas on multiple length scales. The SAXS analysis code is applicable to any two-phase system and is freely available to the public. The total surface area determined by SAXS was 12% greater than that obtained by gas adsorption. The macropore interfacial area, however, is only 30% of the external surface area determined by the αs-plot. The overestimation of the external surface area by the αs-plot method is attributed to capillary condensation in nanoscale surface irregularities. The discrepancy is resolved assuming that the macropore-globule interfaces harbor fractally distributed nooks and crannies, which lead to gas adsorption at pressures above the mesopore filling pressure.

Inherent flexibility of CLIC6 revealed by crystallographic and solution studies

Chloride intracellular channels (CLICs) are a family of unique proteins, that were suggested to adopt both soluble and membrane-associated forms. Moreover, following this unusual metamorphic change, CLICs were shown to incorporate into membranes and mediate ion conduction in vitro, suggesting multimerization upon membrane insertion. Here, we present a 1.8 Å resolution crystal structure of the CLIC domain of mouse CLIC6 (mCLIC6). The structure reveals a monomeric arrangement and shows a high degree of structural conservation with other CLICs. Small-angle X-ray scattering (SAXS) analysis of mCLIC6 demonstrated that the overall solution structure is similar to the crystallographic conformation. Strikingly, further analysis of the SAXS data using ensemble optimization method unveiled additional elongated conformations, elucidating high structural plasticity as an inherent property of the protein. Moreover, structure-guided perturbation of the inter-domain interface by mutagenesis resulted in a population shift towards elongated conformations of mCLIC6. Additionally, we demonstrate that oxidative conditions induce an increase in mCLIC6 hydrophobicity along with mild oligomerization, which was enhanced by the presence of membrane mimetics. Together, these results provide mechanistic insights into the metamorphic nature of mCLIC6.

Expanded Kinetic Control for Persistent Micelle Templates with Solvent Selection

The precision control of nanoscale materials remains a challenge for the study of nanostructure-performance relationships. Persistent micelle templates (PMT) are a kinetic-controlled self-assembly approach that decouples pore and wall control. Here, block copolymer surfactants form persistent micelles that maintain constant template size as material precursors are added, despite the shifting equilibrium dimensions. Earlier PMT demonstrations were based upon solvent mixtures where kinetic rates were adjusted with the amount of water cosolvent. This approach is however limited because ever-higher water contents can lead to secondary porosity within the material walls. Herein, we report an improved method to regulate the PMT kinetics via the majority solvent. This enables a new avenue for the expansion of the PMT window to realize templated materials with a greater extent of tunability. In addition, we report a new small-angle X-ray scattering (SAXS)-based log-log analysis method to independently test the micelle-templated series for consistency with the expected lattice expansion with an increasing material:template ratio. The PMT window identified by the log-log analysis of the SAXS data agreed well with independent scanning electron microscopy measurements. The combination of improved micelle control with solvent selection along with SAXS validation will accelerate the development of a myriad of nanomaterial applications.

Temperature-Dependent Atomic Models of Detergent Micelles Refined against Small-Angle X-Ray Scattering Data.

Surfactants have found a wide range of industrial and scientific applications. In particular, detergent micelles are used as lipid membrane mimics to solubilize membrane proteins for functional and structural characterization. However, an atomic-level understanding of surfactants remains limited because many experiments provide only low-resolution structural information on surfactant aggregates. In this work, small-angle X-ray scattering is combined with molecular dynamics simulations to derive fully atomic models of two maltoside micelles at temperatures between 10 °C and 70 °C. The micelles take the shape of general tri-axial ellipsoids and decrease in size and aggregation number with increasing temperature. Density profiles of hydrophobic groups and water along the three principal axes reveal that the minor micelle axis closely mimics lipid membranes. The results suggest that coupling atomic simulations with low-resolution data allows the structural characterization of surfactant aggregates.

Structural characterization of multilamellar liposomes coencapsulating curcumin and vitamin D3

This study aimed the production of multilamellar liposomes coencapsulating two hydrophobic bioactives, curcumin and vitamin D3, which present antioxidant and nutraceutical properties. Liposomes were produced by proliposomes hydration and stabilized with a mixture of xanthan and guar gums. Proliposomes were produced by coating of micronized sucrose method. Physicochemical properties of the dispersions were evaluated over 42 days of refrigerated storage. Microstructural characterization were performed by differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and small deformation rheological analyses. Liposomes showed spherical and near-spherical shapes and presented hydrodynamic diameters in the 680–1580 nm range. Curcumin and cholecalciferol were loaded into the same vesicles, with preservation efficiencies ranging from 89.6 to 93.3% and from 88.3 to 91.3%, respectively, after 42 days of storage. SAXS data showed that the lipid bilayer of liposomes produced in the presence of curcumin, vitamin D3, or a mixture of both, are very similar. Also, higher amounts of vitamin D3 did not affect negatively the stability of phospholipid vesicles. Overall, it was demonstrated that the coencapsulation of curcumin and vitamin D3 by liposomes is feasible and high contents of bioactives were retained throughout the storage time.

Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12.

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography-SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

Determination of protein oligomeric structure from small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self-association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.

Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy.

Experimental data offers empowering constraints for structure prediction. These constraints can be used to filter equivalently scored models or more powerfully within optimization functions toward prediction. In CASP12, Small Angle X-ray Scattering (SAXS) and Cross-Linking Mass Spectrometry (CLMS) data, measured on an exemplary set of novel fold targets, were provided to the CASP community of protein structure predictors. As solution-based techniques, SAXS and CLMS can efficiently measure states of the full-length sequence in its native solution conformation and assembly. However, this experimental data did not substantially improve prediction accuracy judged by fits to crystallographic models. One issue, beyond intrinsic limitations of the algorithms, was a disconnect between crystal structures and solution-based measurements. Our analyses show that many targets had substantial percentages of disordered regions (up to 40%) or were multimeric or both. Thus, solution measurements of flexibility and assembly support variations that may confound prediction algorithms trained on crystallographic data and expecting globular fully-folded monomeric proteins. Here, we consider the CLMS and SAXS data collected, the information in these solution measurements, and the challenges in incorporating them into computational prediction. As improvement opportunities were only partly realized in CASP12, we provide guidance on how data from the full-length biological unit and the solution state can better aid prediction of the folded monomer or subunit. We furthermore describe strategic integrations of solution measurements with computational prediction programs with the aim of substantially improving foundational knowledge and the accuracy of computational algorithms for biologically-relevant structure predictions for proteins in solution.

Enhanced Sampling of LNCRNA Conformational Space for Defining Ensembles of Structures Used in Ensemble Docking and Virtual Screening of RNA-Focused Small Molecules

RNA remains an underexplored class of drug targets owing to the limited number of RNA-focused experimental and computational screening assays. Importantly, the highly charged phosphate backbone and inherent flexibility unique to RNA macromolecules present computational challenges to virtual screening techniques involving conformational sampling and molecular docking. We have developed a computational methodology to examine docking of small molecules to an ensemble of RNA conformations. A pipeline is developed to target a critical triple helix in the oncogenic MALAT1, a long noncoding RNA that promotes metastasis in a large number of cancer types. We use a conformational FRET assay to show that the MALAT1 triple helix (M1TH) samples a large conformational space consisting of both native states at physiological conditions and inhibitory states when solutions conditions are altered. Preliminary Small Angle X-ray Scattering (SAXS) experiments show a change in radius of gyration (Rg) consistent with conformational FRET data. Replica Exchange Molecular Dynamics are being used to calculate two ensemble sets (i) native-like conformations and (ii) inhibitory conformations) of M1TH structures. Structural states are clustered based on Rg consistent with SAXS data. The relatively small size of the 76 nucleotide M1TH simulation structure allows rigorous sampling of conformational space. We hypothesize that small molecule induced disruption of this RNA triple helix is a novel approach for therapeutic intervention to inhibit progression of multiple cancer types. M1TH ensembles will be used in ensemble docking of compounds identified through a shape-based virtual screening methodology to identify novel M1TH-specific lead compounds that reduce cancer proliferation.

Assessment of collagen fiber orientation dispersion in articular cartilage by small-angle X-ray scattering and diffusion tensor imaging: Preliminary results

Measurements of the orientational dispersion of collagen fibers in articular cartilage were made using diffusion tensor imaging (DTI) and small-angle X-ray scattering (SAXS) on matched bovine articular cartilage samples. Thirteen pairs of samples were excised from bovine knee joints; each pair was taken from neighboring locations in the same bone. One sample from each pair was used for DTI measurements and the other for SAXS measurements. Fractional anisotropy (FA) values were calculated from the DTI data both for the individual imaging voxels and for whole regions of interest (ROI). The FA values were used as a measure of fiber dispersion and compared to the ellipticities of the fiber orientation distributions obtained from SAXS. Neither the spatially-resolved FA values nor whole-ROI FA values showed any correlation with SAXS ellipticities. We attribute the lack of DTI-SAXS correlation to two principal factors: (1) the significant difference in the imaging resolution of the two techniques; and (2) the inherent limitations of both the SAXS data analysis methodology and the diffusion tensor model in the case of multi-modal fiber orientation distributions. We discuss how these factors could be overcome in future work.

Hybrid Methods for Modeling Protein Structures Using Molecular Dynamics Simulations and Small-Angle X-Ray Scattering Data.

Small-angle X-ray scattering (SAXS) is an efficient experimental tool to measure the overall shape of macromolecular structures in solution. However, due to the low resolution of SAXS data, high-resolution data obtained from X-ray crystallography or NMR and computational methods such as molecular dynamics (MD) simulations are complementary to SAXS data for understanding protein functions based on their structures at atomic resolution. Because MD simulations provide a physicochemically proper structural ensemble for flexible proteins in solution and a precise description of solvent effects, the hybrid analysis of SAXS and MD simulations is a promising method to estimate reasonable solution structures and structural ensembles in solution. Here, we review typical and useful in silico methods for modeling three dimensional protein structures, calculating theoretical SAXS profiles, and analyzing ensemble structures consistent with experimental SAXS profiles. We also review two examples of the hybrid analysis, termed MD-SAXS method in which MD simulations are carried out without any knowledge of experimental SAXS data, and the experimental SAXS data are used only to assess the consistency of the solution model from MD simulations with those observed in experiments. One example is an investigation of the intrinsic dynamics of EcoO109I using the computational method to obtain a theoretical profile from the trajectory of an MD simulation. The other example is a structural investigation of the vitamin D receptor ligand-binding domain using snapshots generated by MD simulations and assessment of the snapshots by experimental SAXS data.

Изучение сверхструктуры в сильно легированном пористом фосфиде индия рентгеновскими методами

AbstractAn indium-phosphide InP sample subjected to the pore-generation procedure and then doped with S atoms is studied by the methods of X-ray diffraction analysis (XRD) and small-angle X-ray scattering (SAXS) (with Cu K _α1-radiation). The XRD data demonstrate that the sample consists of (coherent) aligned homogeneous components. A point detector is used to obtain, in the anomalous transmission mode by Borrmann, a set of SAXS curves at sample positions varied by azimuthal rotations. The SAXS data are used to simulate a 2D SAXS pattern for the sample under study, which makes it possible to determine the long-distance translation symmetry and, consequently, the presence of a superstructure. The interplanar distances in the superstructure in the directions (110) and (1 0) of the InP lattice are found to be ~260 and 450 nm, respectively. The symmetry group of the superstructure is determined as C _2 v in the (001) plane of the sample lattice.

X-Ray Study of the Superstructure in Heavily Doped Porous Indium Phosphide

An indium-phosphide InP sample subjected to the pore-generation procedure and then doped with S atoms is studied by the methods of X-ray diffraction analysis (XRD) and small-angle X-ray scattering (SAXS) (with CuKα1-radiation). The XRD data demonstrate that the sample consists of (coherent) aligned homogeneous components. A point detector is used to obtain, in the anomalous transmission mode by Borrmann, a set of SAXS curves at sample positions varied by azimuthal rotations. The SAXS data are used to simulate a 2D SAXS pattern for the sample under study, which makes it possible to determine the long-distance translation symmetry and, consequently, the presence of a superstructure. The interplanar distances in the superstructure in the directions (110) and (1 0) of the InP lattice are found to be ~260 and 450 nm, respectively. The symmetry group of the superstructure is determined as C2v in the (001) plane of the sample lattice.

Noninvasive Structural Analysis of Intermediate Species During Fibrillation: An Application of Small-Angle X-Ray Scattering.

Structural investigation of intermediately formed oligomers and pre-fibrillar species is of tremendous importance in order to elucidate the structural principles of fibrillation, and because intermediate species have been suggested as the pathogenic agents in several amyloid diseases. Structural investigations are however greatly complicated by the dynamic changesbetween structural states of very different sizes and life-times. Small angle X-ray scattering (SAXS) is an ideal method to handle this challenge. The method provides information about the fibrillation process (number of species present and their volume fractions) and low-resolution 3-dimensional structural models of individual species, notably also of the intermediately formed, in-process species from undisturbed fibrillation equilibria. Here, we provide a detailed description of the methods used for the measurement and analysis of SAXS data from fibrillating samples, exemplified using data from our own research.

Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway.

Small-angle X-ray scattering (SAXS) is an invaluable complement to other biophysical methods used to interrogate the structure and dynamics of proteins. Here, we describe the standard experimental protocol used in our laboratory to analyze proteins in the ubiquitin pathway. The method addresses buffer selection, data collection using an in-house X-ray source, diagnostic tests to assess data quality, and computational approaches to interpret SAXS data.

Ultrastructure of potato starch granules as affected by microwave treatment

ABSTRACTThe effect of microwave heating on the lamellar ultrastructure of potato starch granules was studied using small-angle X-ray scattering (SAXS), and the linear correlation function was generated from SAXS data to obtain the parameters of the lamellar repeats architecture. To investigate how the molecular vibration of the microwaves influences the lamellar structure during heating, rapid convection heating was applied through an oil bath for comparison. The results showed that the samples subjected to the microwave and the rapid convection heating displayed similar data, which could demonstrate that the effect of microwave heating on submicroscopic structure of potato starch may be mainly due to its rapid heating effect.