Experimental Small-angle X-ray Scattering Research Articles

Combining small angle X-ray scattering (SAXS) with protein structure predictions to characterize conformations in solution.

Accurate protein structure predictions, enabled by recent advances in machine learning algorithms, provide an entry point to probing structural mechanisms and to integrating and querying many types of biochemical and biophysical results. Limitations in such protein structure predictions can be reduced and addressed through comparison to experimental Small Angle X-ray Scattering (SAXS) data that provides protein structural information in solution. SAXS data can not only validate computational predictions, but can improve conformational and assembly prediction to produce atomic models that are consistent with solution data and biologically relevant states. Here, we describe how to obtain protein structure predictions, compare them to experimental SAXS data and improve models to reflect experimental information from SAXS data. Furthermore, we consider the potential for such experimentally-validated protein structure predictions to broadly improve functional annotation in proteins identified in metagenomics and to identify functional clustering on conserved sites despite low sequence homology.

Structural analysis of a calix[4]arene-based Platonic Micelle

We have recently introduced the concept of “Platonic micelles”, the preference of spherical micelles to specific aggregation numbers mostly coinciding with the number of faces of platonic solids. This effect was observed on bulky, mostly calix[4]arene-based surfactant systems with small aggregation numbers. The preferred aggregation numbers result in better sphere coverage, highliting the packing and the “protection” of hydrophobic cores from the aqueous solvent as the most important factor for this preference. In the present study we further explore the interactions that drive the packing of the highly charged PACaL3 surfactant into highly symmetrical hexameric micelles. We performed a series of molecular dynamics simulations that yielded a large set of structures and an ensemble in good agreement with the experimental Small Angle X-ray Scattering data was selected. The geometry and the rigidity of the calix[4]arene group with proper tail length and headgroup volume are the driving forces for the high symmetry and monodispersity of the micelle. The charge of the headgroups is mainly responsible for inhibiting the formation of higher order structures. Sodium, shown to be important for the stability of the micelle, is not directly interacting with the micelle implying that the calix[4]arene ring is a C2ν symmetry conformation.

Mesoscale pore structure of a high‐performance concrete by coupling focused ion beam/scanning electron microscopy and small angle X‐ray scattering

AbstractThis contribution couples (a) Small angle X‐ray scattering (SAXS) experiments of a high‐performance concrete (HPC) at the millimetric scale, and (b) Focused ion beam/scanning electron microscopy (FIB/SEM) of the cement paste of the HPC, with 10‐20 nm voxel size. The aim is to improve the understanding of the 3D pore network of the HPC at the mesoscale (tens of nm), which is relevant for fluid transport. The mature HPC is an industrial concrete, based on pure Portland CEMI cement, and planned for use as structural elements for deep underground nuclear waste storage. Small angle X‐ray scattering patterns are computed from the 3D pore images given by FIB/SEM (volumes of 61‐118 μm3). They are positively correlated with SAXS measurements (volumes of 5 mm3). Aside from correlations with FIB/SEM data, experimental SAXS allows to investigate a wider range of effects on the pore structure. These are mainly the HPC drying state, the presence of aggregates (by analyzing data on cement paste alone), and the use of Poly Methyl MethAcrylate resin impregnation.

Rod-like particles growing in sol–gel processing of 1:1 molar mixtures of 3-glycidoxypropyltrimethoxysilane and tetraethoxysilane

The growth kinetics and the structure of organic/silica hybrids prepared from acid hydrolysis of 1:1 molar mixtures of 3-glycidoxypropyltrimethoxysilane and tetraethoxysilane were studied by small-angle X-ray scattering (SAXS) at 315, 325 and 335 K. The evolution of the SAXS intensity is compatible with the growth of silica-rich domains by aggregation from a fixed number of primary particles. Two distinct growth regimes could be identified by analyzing the relation I(0) ∝ R g D between the intensity extrapolated to zero I(0), the radius of gyration R g of the aggregates and the exponent D, which gives information on the geometry and the mechanism of growth of the aggregates. An initial period was attributed to the growth of rod-like particles with approximately the same radius and variable length. At more advanced degrees of aggregation the process was controlled by the growth of larger aggregates with higher-order dimensionality. A narrow distribution of cylinder lengths given by the Schulz function fitted the experimental SAXS data well during the most part of the initial regime of cylindrical particle growth. These particles were later found as rod-like subunits of the larger aggregates grown at more advanced degrees of aggregation. Some condensation possibilities yielding the formation of structures compatible with those inferred from the present study are discussed.

Coarse-Grained Molecular Modeling of the Solution Structure Ensemble of Dengue Virus Nonstructural Protein 5 with Small-Angle X-ray Scattering Intensity.

An ensemble-modeling scheme incorporating coarse-grained simulations with experimental small-angle X-ray scattering (SAXS) data is applied to dengue virus 2 (DENV2) nonstructural protein 5 (NS5). NS5 serves a key role in viral replication through its two domains that are connected by a 10-residue polypeptide segment. A set of representative structures is generated from a simulated structure pool using SAXS data fitting by the non-negativity least squares (NNLS) or standard ensemble optimization method (EOM) based on a genetic algorithm (GA). It is found that a proper low-energy threshold of the structure pool is necessary to produce a conformational ensemble of two representative structures by both NNLS and GA that agrees well with the experimental SAXS profile. The stability of the constructed ensemble is validated also by molecular dynamics simulations with an all-atom force field. The constructed ensemble successfully revealed the domain-domain orientation and domain-contacting interface of DENV2 NS5. Using experimental data fitting and additional investigations with synthesized data, it is found that energy restraint on the conformational pool is necessary to avoid overinterpretation of experimental data by spurious conformational representations.

Probing Manganese Ion Distributions Around Nucleic Acids using Small Angle X-Ray Scattering

Manganese ions are divalent cations that have profound biological roles in cellular processes, e.g. they are involved in folding of some RNAs and in some DNA conformational changes. We use two experimental Small-Angle X-ray Scattering (SAXS) based approaches to probe the distribution of Manganese ions around nucleic acids. SAXS is a solution-based technique that is sensitive to the ion cloud and hydration layer around nucleic acids. By absolutely calibrating SAXS data, we can accurately characterize the ion cloud distribution and the water layer around DNA.

Structural Properties of Protein-Detergent Complexes from SAXS and MD Simulations.

In experimental studies of solubilized membrane proteins, the detergent corona influences the protein behavior and the resulting measurement. Thus, combinations of experimental techniques with atomistic modeling have been used to resolve corona structural parameters and distributions. Here, we used small-angle X-ray scattering (SAXS) data and molecular dynamics simulations to study a model protein-detergent complex (PDC) consisting of aquaporin-0 and dodecyl-β-maltoside molecules (βDDM). The corona morphology of single snapshots was found to be rough, but it is smooth and compacted in 100-ns-scale ensemble averages. Individual snapshots therefore were unable to accurately represent the ensemble information as captured by experimental SAXS. Mimicking of annular lipids by detergent was also observed. SAXS prediction using different published methods was used to identify optimal βDDM numbers. Explicit-solvent methods predicted best agreement using 290-βDDM PDCs, but implicit-solvent methods gave unclear predictions due to overcompensation by free solvation-layer density parameters. Thus, ensemble-based approaches and physically motivated constraints will help to extract structural information from SAXS data.

Large-scale motions in the adenylate kinase solution ensemble: Coarse-grained simulations and comparison with solution X-ray scattering

While coarse-grained (CG) simulations provide an efficient approach to identify small- and large-scale motions important to protein conformational transitions, coupling with appropriate experimental validation is essential. Here, by comparing small-angle X-ray scattering (SAXS) predictions from CG simulation ensembles of adenylate kinase (AK) with a range of energetic parameters, we demonstrate that AK is flexible in solution in the absence of ligand and that a small population of the closed form exists without ligand. In addition, by analyzing variation of scattering patterns within CG simulation ensembles, we reveal that rigid-body motion of the LID domain corresponds to a dominant scattering feature. Thus, we have developed a novel approach for three-dimensional structural interpretation of SAXS data. Finally, we demonstrate that the agreement between predicted and experimental SAXS can be improved by increasing the simulation temperature or by computationally mutating selected residues to glycine, both of which perturb LID rigid-body flexibility.

Size Distribution of Gold Nanoparticles Used by Small Angle X-ray Scattering

ABSTRACT The size distribution of gold nanoparticles was estimated based on the scattering intensity data obtained from small angle X-ray scattering (SAXS) and compared with the result of micrographs by transmission electron microscopy. The slope of the Guinier plot was used to estimate the mean size assuming a narrow particle size distribution. When the size distribution is narrow, the mean size can be easily obtained from slope of a Guinier plot of scattering data within 10% error. Additionally, assuming a lognormal size distribution, the size distribution and the mean size can be calculated using the experimental SAXS data in the fitting analysis. The histogram method, which utilizes the coefficient matrix of scattering intensity, was also applied to the estimation of the size distribution, and this method could be useful for a rough estimate of the size distribution.

Insight on Structural Change in Sol?Gel-Derived Silica Gel with Aging under Basic Conditions for Mesopore Control

Structural rearrangement of sol–gel-derived silica gel by aging under basic conditions was investigated using small angle X-ray scattering (SAXS) and 29Si nuclear magnetic resonance (NMR). A wet silica gel prepared under acidic conditions had a fractal nature, and many unreacted silanols remained on the surface. During aging of the gel in ammonia solution, additional Si—O—Si bonds rapidly formed, whereas the change in mesoscale structure gradually proceeded. This result was compared with that of simulation modeling the Ostwald ripening, i.e. dissolution from positive curvature and reprecipitation on negative curvature. In the simulation, structural change in a cluster from fractal nature to particle aggregates was well visualized in 2-dimmensional square lattice. Both scattering profiles calculated from the model clusters and the change in average coordination number of monomers in the cluster well agreed with the experimental SAXS and NMR results, respectively. This agreement strongly ensures us that structural change by aging under basic conditions proceeds through the Ostwald ripening. The mesopore size as well as mesopore volume in calcined silica gel is determined by the shrinkage degree during drying and calcination. The sample aged in basic solution restrains the shrinkage because of the growth of particulate structure, and retains large size and volume of pores.

An improved small-angle X-ray scattering analysis of δ′ precipitation in Al-Li alloy with hard-sphere interaction

Al-Li alloys are of particular interest for the aircraft industry, due to their low density and high strength. Precipitation of the alloy results in hardening of the material, which is of great technological importance. During the coarsening stage of {delta}{prime} precipitation the particles have a wide size distribution (polydisperse form). The interparticle interference effect in the small-angle X-ray scattering (SAXS) intensities arose from the high volume fraction of {delta}{prime} precipitates, and it makes the SAXS analysis much more difficult. So recent SAXS studies for Al-Li alloys still analyze the SAXS data by using either average radius (monodisperse form) or presumed size distribution, and a hard-sphere interaction model for the {delta}{prime} particle interference effect. In the work, an improved algorithm is developed to reconstruct the particle size distribution from SAXS intensities without any prior assumed size distribution. The hard-sphere interaction effect is also combined in the calculation. This method will be tested by analyzing simulated SAXS data and show an advantage in comparison with the other method. Then, it is used to reconstruct the particle size distribution from SAXS intensities without any prior assumed size distribution. The hard-sphere interaction effect is also combined in the calculation. This method will be testedmore » by analyzing simulated SAXS data and show an advantage in comparison with the other method. Then, it is used to reconstruct the particle size distribution of {delta}{prime} precipitates by analyzing a set of experimental SAXS data from Al-9.7 at% Li alloy having strong correlation peak, which results from inter-particle interference effect. More accurate parameters about structure and thermodynamic information can be extracted by employing the new method. The results of the analysis are also compared with the asymmetric size distribution model given by a growth kinetics theory, called modified Lifshiftz-Slyozov-Wagner (MLSW) theory.« less

Alternative model for the fine porous structure in hydrated tricalcium silicate

Synopsis Hydrated tricalcium silicate has been studied by small angle X-ray scattering (SAXS). The experimental SAXS results have been compared with those expected from a lamellar model and from the Debye, Anderson and Brumberger (DAB) model. The theoretical SAXS intensity for the DAB structural model agrees closely with the experimental results. In addition the DAB model provides a good explanation of the different values of specific surface area determined by SAXS, adsorption and proton NMR methods.