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Concept: Small-angle X-ray scattering


Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV[Formula: see text]. ATV[Formula: see text] forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV[Formula: see text] displays a new [Formula: see text] protein fold, consistent with the absence of homologues of this protein in public sequence databases.

Concepts: DNA, Protein, Gene, Molecular biology, Virus, Chemistry, X-rays, Small-angle X-ray scattering


Water exists in high- and low-density amorphous ice forms (HDA and LDA), which could correspond to the glassy states of high- (HDL) and low-density liquid (LDL) in the metastable part of the phase diagram. However, the nature of both the glass transition and the high-to-low-density transition are debated and new experimental evidence is needed. Here we combine wide-angle X-ray scattering (WAXS) with X-ray photon-correlation spectroscopy (XPCS) in the small-angle X-ray scattering (SAXS) geometry to probe both the structural and dynamical properties during the high-to-low-density transition in amorphous ice at 1 bar. By analyzing the structure factor and the radial distribution function, the coexistence of two structurally distinct domains is observed at T = 125 K. XPCS probes the dynamics in momentum space, which in the SAXS geometry reflects structural relaxation on the nanometer length scale. The dynamics of HDA are characterized by a slow component with a large time constant, arising from viscoelastic relaxation and stress release from nanometer-sized heterogeneities. Above 110 K a faster, strongly temperature-dependent component appears, with momentum transfer dependence pointing toward nanoscale diffusion. This dynamical component slows down after transition into the low-density form at 130 K, but remains diffusive. The diffusive character of both the high- and low-density forms is discussed among different interpretations and the results are most consistent with the hypothesis of a liquid-liquid transition in the ultraviscous regime.

Concepts: Ice, Force, Viscosity, Phase transition, Glass, Glass transition, X-rays, Small-angle X-ray scattering


In metabolic bone diseases, the alterations in fibrillar level bone-material quality affecting macroscopic mechanical competence are not well-understood quantitatively. Here, we quantify the fibrillar level deformation in cantilever bending in a mouse model for hereditary rickets (Hpr). Microfocus in-situ synchrotron small-angle X-ray scattering (SAXS) combined with cantilever bending was used to resolve nanoscale fibril strain in tensile- and compressive tissue regions separately, with quantitative backscattered scanning electron microscopy used to measure microscale mineralization. Tissue-level flexural moduli for Hpr mice were significantly (p<0.01) smaller compared to wild-type (~5 to 10-fold reduction). At the fibrillar level, the fibril moduli within the tensile and compressive zones were significantly (p<0.05) lower by ~3- to 5-fold in Hpr mice compared to wild-type mice. Hpr mice have a lower mineral content (24.2±2.1Cawt.% versus 27.4±3.3Ca wt.%) and its distribution was more heterogeneous compared to wild-type animals. However, the average effective fibril modulus did not differ significantly (p>0.05) over ages (4, 7 and 10weeks) between tensile and compressive zones. Our results indicate that incompletely mineralized fibrils in Hpr mice have greater deformability and lower moduli in both compression and tension, and those compressive and tensile zones have similar moduli at the fibrillar level.

Concepts: Electron, Dietary mineral, Mineral, X-ray crystallography, Scanning electron microscope, Young's modulus, X-rays, Small-angle X-ray scattering


As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abelé’s matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.

Concepts: Cell membrane, Scattering, Scientific techniques, Angle, Lipid bilayer, X-rays, Biological small-angle scattering, Small-angle X-ray scattering


In liquid-liquid extraction, organic phase splitting arises when high concentrations of polar solutes (acids/metal ions) are extracted. Herein, we investigate the mesoscopic roots that underpin phase splitting in alkane phases containing mixed amphiphiles, of contemporary interest in solvent extraction separation systems, by extracting various oxoacids. The oxoacids exhibited individual macroscopic (extractive and physical) behaviors, inducing phase splitting into heavy and light domains under markedly different conditions. Using small-angle X-ray scattering (SAXS) data analyzed using the generalized indirect Fourier transform (GIFT) method, we showed that, in all cases, acid extraction drove the self-assembly of reverse micelles into rods. These grew with increased acid extraction until reaching a critical length of 20 nm, at which point interactions produced interconnected cylinders or lamellar sheets that prelude phase splitting into heavy and light domains. In all cases, the heavy phase contained the same surfactant ratio-TBP (tri-n-butyl phosphate) and CMPO (octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide)-even though the concentrations of acid, water, and amphiphiles were markedly different. The remarkable similarities in structure and amphiphile stoichiometries underpinning phase splitting across the macroscopically different acid extraction series allude to the mesoscopic roots of organic phase behavior in solvent extraction. Our studies show that the structures underpinning phase splitting in solvent extraction systems are more complex than previously thought and are reminiscent of phase transitions in soft matter.

Concepts: Fundamental physics concepts, Solubility, Fourier transform, Solution, Small-angle X-ray scattering, Liquid-liquid extraction, Acid-base extraction, Tributyl phosphate


In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5°) and wide angles (typically > 5°). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg’s law and is dependent on the wavelength and incident angle. In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm. Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

Concepts: Diffraction, Chemistry, Polymer, X-ray crystallography, Macromolecule, William Lawrence Bragg, X-rays, Small-angle X-ray scattering


When used in combination with raster scanning, small-angle X-ray scattering (SAXS) has proven to be a valuable imaging technique of the nanoscale, for example of bone, teeth and brain matter. Although two-dimensional projection imaging has been used to characterize various materials successfully, its three-dimensional extension, SAXS computed tomography, poses substantial challenges, which have yet to be overcome. Previous work using SAXS computed tomography was unable to preserve oriented SAXS signals during reconstruction. Here we present a solution to this problem and obtain a complete SAXS computed tomography, which preserves oriented scattering information. By introducing virtual tomography axes, we take advantage of the two-dimensional SAXS information recorded on an area detector and use it to reconstruct the full three-dimensional scattering distribution in reciprocal space for each voxel of the three-dimensional object in real space. The presented method could be of interest for a combined six-dimensional real and reciprocal space characterization of mesoscopic materials with hierarchically structured features with length scales ranging from a few nanometres to a few millimetres-for example, biomaterials such as bone or teeth, or functional materials such as fuel-cell or battery components.

Concepts: Diffraction, Medical imaging, Dimension, Vector space, Object-oriented programming, X-rays, Small-angle X-ray scattering, Synchrotron related techniques


Microtubules (MTs) are dynamic structures that are fundamental for cell morphogenesis and motility. MT-associated motors work efficiently to perform their functions. Unlike other motile kinesins, KIF2 catalytically depolymerizes MTs from the peeled protofilament end during ATP hydrolysis. However, the detailed mechanism by which KIF2 drives processive MT depolymerization remains unknown. To elucidate the catalytic mechanism, the transitional KIF2-tubulin complex during MT depolymerization was analyzed through multiple methods, including atomic force microscopy, size-exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering, analytical ultracentrifugation, and mass spectrometry. The analyses outlined the conformation in which one KIF2core domain binds tightly to two tubulin dimers in the middle pre-hydrolysis state during ATP hydrolysis, a process critical for catalytic MT depolymerization. The X-ray crystallographic structure of the KIF2core domain displays the activated conformation that sustains the large KIF2-tubulin 1:2 complex.

Concepts: Protein, Adenosine triphosphate, Eukaryote, Scattering, Kinesin, Tubulin, Microtubule, Small-angle X-ray scattering


The nanoscale structure of milk fat (MF) crystal networks is extensively described for the first time through the characterization of milk fat-crystalline nanoplatelets (MF-CNPs). Removing oil by washing with cold isobutanol and breaking-down crystal aggregates by controlled homogenization allowed for the extraction and visualization of individual MF-CNPs that are mainly composed of high melting triacylglycerols (TAGs). By image analysis, the length and width of MF-CNPs were measured (600 nm × 200 nm-900 nm × 300 nm). Using small-angle X-ray scattering (SAXS), crystalline domain size, (i.e., thickness of MF-CNPs), was determined (27 nm (d001)). Through interpretation of ultra-small-angle X-ray scattering (USAXS) patterns of MF using Unified Fit and Guinier-Porod models, structural properties of MF-CNPs (smooth surfaces) and MF-CNP aggregations were characterized (RLCA aggregation of MF-CNPs to form larger structures that present diffused surfaces). Elucidation of MF-CNPs provides a new dimension of analysis for describing MF crystal networks and opens-up opportunities for modifying MF properties through nanoengineering.

Concepts: Structure, Milk, Materials science, Height, X-rays, Small-angle X-ray scattering, Small-angle scattering, Cream


Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, dithiothreitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies.

Concepts: Concentration, Chemistry, Scattering, Liquid, Surface tension, Molar concentration, X-rays, Small-angle X-ray scattering