Newly developed Laboratory-based Size exclusion chromatography Small-angle x-ray scattering System (La-SSS)

Inoue, Rintaro; Nakagawa, Tatsuo; Morishima, Ken; Sato, Nobuhiro; Okuda, Aya; Urade, Reiko; Yogo, Rina; Yanaka, Saeko; Yagi-Utsumi, Maho; Kato, Koichi; Omoto, Kazuki; Ito, Kazuki; Sugiyama, Masaaki

doi:10.1038/s41598-019-48911-w

Cited by 22 publications

(15 citation statements)

References 16 publications

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“…1), which in turn has spurred developments in sample environments and instrument hardware (Classen et al, 2013;Kirby et al, 2013;Pernot et al, 2013;Acerbo et al, 2015;Blanchet et al, 2015;Heller et al, 2018;Liu et al, 2018;Wood et al, 2018). For instance, size-exclusion chromatography coupled to SAS (SEC-SAS), first demonstrated by Mathew et al (2004), has now become a routine approach for the separation and structural analysis of mixture components, and is offered at many SAXS and SANS beamlines, as well as for laboratory instruments (David & Pé rez, 2009;Graewert et al, 2015;Jordan et al, 2016;Brennich et al, 2017;Yeh et al, 2017;Johansen et al, 2018;Ryan et al, 2018;Bucciarelli et al, 2018;Inoue et al, 2019). Improved X-ray detectors enable timeresolved measurements at shorter timescales and, in combination with lasers and rapid mixing devices, facilitate the study of macromolecular kinetics (Cammarata et al, 2008;Kubelka, 2009;Pollack, 2011;Graceffa et al, 2013;Levantino et al, 2015;Tuukkanen et al, 2017;Josts et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis

et al. 2021

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The ATSAS software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the ATSAS 3.0 package are described. They include IMSIM, for simulating isotropic 2D scattering patterns; IMOP, to perform operations on 2D images and masks; DATRESAMPLE, a method for variance estimation of structural invariants through parametric resampling; DATFT, which computes the pair distance distribution function by a direct Fourier transform of the scattering data; PDDFFIT, to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in DATMW for Bayesian consensus-based concentration-independent molecular weight estimation; DATMIF, an ab initio shape analysis method that optimizes the search model directly against the scattering data; DAMEMB, an application to set up the initial search volume for multiphase modelling of membrane proteins; ELLLIP, to perform quasi-atomistic modelling of liposomes with elliptical shapes; NMATOR, which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; DAMMIX, which reconstructs the shape of an unknown intermediate in an evolving system; and LIPMIX and BILMIX, for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the PRIMUS graphical interface to Qt5, updating SASpy – a PyMOL plugin to run a subset of ATSAS tools – to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future ATSAS releases. All these features are implemented in ATSAS 3.0, freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.

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Section: Introductionmentioning

confidence: 99%

ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis

et al. 2021

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“…To reduce contamination and aggregates in the samples for SAXS measurements, SEC-SAXS was applied to the samples. In this measurement, a stopping mode in a laboratory-based SEC-SAXS system was adapted to increase the statistics of scattering intensity (see the “ SAXS ” section) 22 .…”

Section: Methodsmentioning

confidence: 99%

“…In the lower- q range measurement (0.01–0.07 Å −1 , SDD = 1330.0 mm), the SEC-SAXS measurement with “stopping mode” 22 was employed to remove the unfavourable aggregates from the sample solution. Then, the scattering data were collected for 1120 min (10 min × 112).…”

Section: Methodsmentioning

confidence: 99%

“…However, SAXS is sensitive to unfavourable contaminations and aggregates in solution, especially large molecules when even in small amounts 18 – 21 . Therefore, we carefully prepared highly purified oxidized and reduced ER-60, examined the quality of the samples, and conducted state-of-the-art SAXS methods combined with size-exclusion chromatography (SEC) (SEC-SAXS) 22 and with analytical ultracentrifugation (AUC) (AUC-SAS) 21 . The integrative organization of these methods allowed us to achieve high-precision aggregation-free SAXS profiles.…”

Section: Introductionmentioning

confidence: 99%

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Solution structure of multi-domain protein ER-60 studied by aggregation-free SAXS and coarse-grained-MD simulation

Okuda

Shimizu

Morishima

et al. 2021

Sci Rep

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Multi-domain proteins (MDPs) show a variety of domain conformations under physiological conditions, regulating their functions through such conformational changes. One of the typical MDPs, ER-60 which is a protein folding enzyme, has a U-shape with four domains and is thought to have different domain conformations in solution depending on the redox state at the active centres of the edge domains. In this work, an aggregation-free small-angle X-ray scattering revealed that the structures of oxidized and reduced ER-60 in solution are different from each other and are also different from those in the crystal. Furthermore, structural modelling with coarse-grained molecular dynamics simulation indicated that the distance between the two edge domains of oxidized ER-60 is longer than that of reduced ER-60. In addition, one of the edge domains has a more flexible conformation than the other.

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“…size-exclusion chromatography SAXS (SEC-SAXS), dynamic light scattering and UV spectrophotometry, have recently been adapted to similar laboratory systems utilizing the powerful flux delivered by the metal-jet technology (Schwamberger et al, 2015;Bucciarelli et al, 2018), enabling the acquisition of simultaneous complementary information while operating the instrument under flow conditions. SEC-SAXS has also recently been done on a rotating-anode laboratory instrument (Inoue et al, 2019). This work also describes procedures for varying the flow to improve the statistics of the SAXS data.…”

Section: Introductionmentioning

confidence: 99%

A high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering

Lyngsø¹,

Pedersen²

2021

J Appl Crystallogr

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A commercially available small-angle X-ray scattering (SAXS) NanoSTAR instrument (Bruker AXS) with a liquid-metal-jet source (Excillum) has been optimized for solution scattering and installed at iNANO at Aarhus University. The instrument (named HyperSAXS) employs long high-quality parabolic Montel multilayer optics (Incoatec) and a novel compact scatterless pinhole slit with Ge edges, which was designed and built at Aarhus University. The combination of the powerful source and optimized geometry gives an integrated X-ray intensity close to 109 photons s−1 for a standard range of scattering vector moduli q = 0.0098–0.425 Å−1, where q = (4πsinθ)/λ and λ is the Ga Kα wavelength of 1.34 Å. The high intensity of the instrument makes it possible to measure dilute samples of, for example, protein or surfactant with concentrations of 1 mg ml−1 in a few minutes. A flow-through cell, built at Aarhus University, in combination with an automated sample handler has been installed on the instrument. The sample handler is based on the commercial Gilson GX-271 injection system (Biolab), which also allows samples to be stored under thermostatted conditions. The sample handler inserts and removes samples, and also cleans and dries the sample cell between measurements. The minimum volume of the flow-through capillary is about 20 µl. The high intensity additionally allows time-resolved measurements to be performed with a temporal resolution of seconds. For this purpose a stopped-flow apparatus, (SFM-3000, Bio-Logic) was connected to the flow-through cell by high-performance liquid chromatography tubing. This configuration was chosen as it allows vacuum around the sample cell and thus maintains a low background. The instrument can readily be converted into a low-q setup with a q range of 0.0049–0.34 Å−1 and an X-ray intensity of about 5 × 107 photons s−1.

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Newly developed Laboratory-based Size exclusion chromatography Small-angle x-ray scattering System (La-SSS)

Cited by 22 publications

References 16 publications

ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis

ATSAS 3.0: expanded functionality and new tools for small-angle scattering data analysis

Solution structure of multi-domain protein ER-60 studied by aggregation-free SAXS and coarse-grained-MD simulation

A high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering

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