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Aggregates cause a fatal problem in the structural analysis of a biomacromolecule in solution using small‐angle X‐ray or neutron scattering (SAS): they deteriorate the scattering profile of the target molecule and lead to an incorrect structure. Recently, an integrated method of analytical ultracentrifugation (AUC) and SAS, abbreviated AUC–SAS, was developed as a new approach to overcome this problem. However, the original version of AUC–SAS does not offer a correct scattering profile of the target molecule when the weight fraction of aggregates is higher than ca 10%. In this study, the obstacle point in the original AUC–SAS approach is identified. The improved AUC–SAS method is then applicable to a solution with a relatively larger weight fraction of aggregates (≤20%). [ABSTRACT FROM AUTHOR]

Understanding protein functions requires not only static but also dynamic structural information. Incoherent quasi-elastic neutron scattering (QENS), which utilizes the highly incoherent scattering ability of hydrogen, is a powerful technique for revealing the dynamics of proteins in deuterium oxide (D2O) buffer solutions. The background scattering of sample cells suitable for aqueous protein solution samples, conducted with a neutron backscattering spectrometer, was evaluated. It was found that the scattering intensity of an aluminum sample cell coated with boehmite using D2O was lower than that of a sample cell coated with regular water (H2O). The D2O-Boehmite coated cell was used for the QENS measurement of a 0.8 wt.% aqueous solution of an intrinsically disordered protein in an intrinsically disordered region of a helicase-associated endonuclease for a fork-structured type of DNA. The cell was inert against aqueous samples at 283–363 K. In addition, meticulous attention to cells with small individual weight differences and the positional reproducibility of the sample cell relative to the spectrometer neutron beam position enabled the accurate subtraction of the scattering profiles of the D2O buffer and the sample container. Consequently, high-quality information on protein dynamics could be extracted from dilute protein solutions. [ABSTRACT FROM AUTHOR]

In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains. The revealed full KaiA12B6C6 complex is assembled including the dynamic and asynchronous KaiA N-terminal domains that have been missing in cryo-EM structures. [ABSTRACT FROM AUTHOR]

Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC–SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins, i.e. a mutated form of IgG-degrading enzyme of Streptococcus pyogenes and a soluble form of Fcγ receptor IIIb, and subjected to SEC–SANS as well as SEC–SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC–iCM–SANS approach. [ABSTRACT FROM AUTHOR]

Recent progress of pulsed neutron sources has enabled energy-resolved analysis of neutron attenuation spectra, which include information on neutron scattering. In this study, a new analysis technique for small-angle neutron scattering (SANS) is demonstrated. A clear difference is observed in the neutron attenuation spectra between steels with different nanostructures; this difference can be understood as arising from attenuation due to SANS. The neutron attenuation spectra calculated from the corresponding SANS profiles agree well with the experimentally observed attenuation spectra. This result indicates that measurement of neutron attenuation spectra may enable the development of a novel experimental technique, i.e. energy-resolved SANS. [ABSTRACT FROM AUTHOR]

Pulsed neutron sources enable the simultaneous measurement of small-angle neutron scattering (SANS) and Bragg edge transmission. This simultaneous measurement is useful for microstructural characterization in steel. Since most steels are ferromagnetic, magnetic scattering contributions should be considered in both SANS and Bragg edge transmission analyses. An expression for the magnetic scattering contribution to Bragg edge transmission analysis has been derived. The analysis using this expression was applied to Cu steel. The ferrite crystallite size estimated from this Bragg edge transmission analysis with the magnetic scattering contribution was larger than that estimated using conventional expressions. This result indicates that magnetic scattering has to be taken into account for quantitative Bragg edge transmission analysis. In the SANS analysis, the ratio of magnetic to nuclear scattering contributions revealed that the precipitates consist of body-centered cubic Cu0.7Fe0.3 and pure Cu, which probably has 9R structure including elastic strain and vacancies. These results show that effective use of the magnetic scattering contribution allows detailed analyses of steel microstructure. [ABSTRACT FROM AUTHOR]