Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SEC-SAXS methods

Zabelskii, Dmitrii; Ryzhykau, Yury L.; Murugova, T. N.; Brennich, Martha; Soloviov, Dmytro; Ivankov, Oleksandr I.; Borshchevskiy, Valentin; Mishin, Alexey; Rogachev, Andrey; Round, Adam; Dencher, Norbert A.; Büldt, Georg; Gordeliy, Valentin; Куклин, А. И.

doi:10.1088/1742-6596/994/1/012017

Cited by 13 publications

(8 citation statements)

References 49 publications

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“…Size-exclusion chromatography coupled with SAS (SEC-SAXS and SEC-SANS techniques) can be used to consider possible oligomerization polydispersity (Mathew et al, 2004;David & Pe ´rez, 2009;Zabelskii et al, 2018;Jordan et al, 2016). In addition, when several curves with variable proportions of oligomers are collected, one can extract each separate curve, as was performed in GASBOR by using the 'mix' mode.…”

Section: Discussionmentioning

confidence: 99%

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ryzhykau¹,

Orekhov²,

Rulev³

et al. 2021

Acta Cryst Sect D Struct Biol

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Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII–NpHtrII) was performed. The influence of the polydispersity of NpSRII–NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

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Section: Discussionmentioning

confidence: 99%

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ryzhykau¹,

Orekhov²,

Rulev³

et al. 2021

Acta Cryst Sect D Struct Biol

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“…For the full-length Np SRII/ Np HtrII and for the truncated complex Np SRII/ Np HtrII 137 , SAXS profiles were obtained for the samples with protein concentrations of 0.57 and 0.78 mg/ml, correspondingly; exposure time was 7 and 10 s, respectively. For all SAXS measurements, peak fractions after gel-filtration were used 68 . See Table S1 for other details of SAXS measurements.…”

Section: Methodsmentioning

confidence: 99%

Molecular model of a sensor of two-component signaling system

Ryzhykau

Orekhov

Rulev

et al. 2021

Sci Rep

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Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.

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“…R g from Region 1 is found to be 53.2 ± 1.6 Å, which is nicely consistent with R g reported by Zabelskii et al . 11 as that of a monomer of AF ( R g = 52.9 Å). On the other hand, R g value satisfying the relationship in Q < 1.3/ R g was not obtained from Region 2 due to too large R g value.…”

Section: Resultsmentioning

confidence: 99%

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

Inoue

Nakagawa²,

Morishima

et al. 2019

Sci Rep

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To understand a biological system, it is important to observe structures of biomolecules in the solution where the system is functionalized. S mall- A ngle X -ray S cattering coupled with S ize E xclusion C hromatography (SEC-SAXS) is one of techniques to selectively observe the target molecules in the multi-component system. However, this technique is believed to be available only with a synchrotron-based SAXS instrument due to requirement of high beam intensity and, therefore, the limitation of the beam time was obstacle to satisfy demands from many bio-researchers. We newly developed La boratory-based S ize exclusion chromatography S AXS S ystem (La-SSS) by utilizing a latest laboratory-based SAXS instrument and finely optimization of the balance between flow rate, cell volume, irradiation time and so on. La-SSS succeeded not only decoupling of target protein(s) from non-specific aggregates but also measurement of each concerned component in a multi-component system. In addition, an option: “stopping mode”, which is designed for improving statistics of SAXS profile, realized a high S/N data acquisition for the most interesting protein in a multi-component system. Furthermore, by utilizing a column having small bed volume, the small-scale SEC-SAXS study makes available. Through optimization of instrumental parameters and environments, La-SSS is highly applicable for experimental requirements from various biological samples. It is strongly expected that a La-SSS concept must be a normal option for laboratory-based SAXS in the near future.

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Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SEC-SAXS methods

Cited by 13 publications

References 49 publications

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Molecular model of a sensor of two-component signaling system

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

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