論文 - 中迫　雅由

Diffraction apparatus and procedure in tomography X-ray diffraction imaging for biological cells at cryogenic temperature using synchrotron X-ray radiation

Kobayashi A., Takayama Y., Okajima K., Oide M., Yamamoto T., Sekiguchi Y., Oroguchi T., Nakasako M., Kohmura Y., Yamamoto M., Hoshi T., Torizuka Y.

Journal of Synchrotron Radiation （Journal of Synchrotron Radiation）  25 （ 6 ） 1803 - 1818 2018年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09090495

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© International Union of Crystallography, 2018 X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 µm at a resolution of 136 nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.

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Classification of ab initio models of proteins restored from small-angle X-ray scattering

Oide M., Sekiguchi Y., Fukuda A., Okajima K., Oroguchi T., Nakasako M.

Journal of Synchrotron Radiation （Journal of Synchrotron Radiation）  25 （ 5 ） 1379 - 1388 2018年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09090495

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© International Union of Crystallography, 2018 In structure analyses of proteins in solution by using small-angle X-ray scattering (SAXS), the molecular models are restored by using ab initio molecular modeling algorithms. There can be variation among restored models owing to the loss of phase information in the scattering profiles, averaging with regard to the orientation of proteins against the direction of the incident X-ray beam, and also conformational fluctuations. In many cases, a representative molecular model is obtained by averaging models restored in a number of ab initio calculations, which possibly provide nonrealistic models inconsistent with the biological and structural information about the target protein. Here, a protocol for classifying predicted models by multivariate analysis to select probable and realistic models is proposed. In the protocol, each structure model is represented as a point in a hyper-dimensional space describing the shape of the model. Principal component analysis followed by the clustering method is applied to visualize the distribution of the points in the hyper-dimensional space. Then, the classification provides an opportunity to exclude nonrealistic models. The feasibility of the protocol was examined through the application to the SAXS profiles of four proteins.

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水和構造に制御される蛋白質のドメイン運動

苙口友隆，中迫雅由

生物物理 58 （ 4 ） 204 - 206 2018年08月

研究論文（学術雑誌）, 共著, 査読有り

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Growth of Cuprous Oxide Particles in Liquid-Phase Synthesis Investigated by X-ray Laser Diffraction

Oroguchi T., Yoshidome T., Yamamoto T., Nakasako M.

Nano Letters （ACS）  18 （ 8 ） 5192 - 5197 2018年07月

共著, 査読有り,  ISSN  15306984

　概要を見る

Copyright © 2018 American Chemical Society. Cuprous oxide (Cu2O) particles obtained by surfactant-assisted liquid-phase synthesis have cuboid shapes but the internal structures are difficult to be visualized by electron microscopy. Herein, we investigated the internal structures of numerous individual Cu2O particles with submicrometer dimensions by X-ray diffraction imaging (XDI) using X-ray free-electron laser (XFEL) pulses. The reconstructed two-dimensional electron density maps, which displayed inhomogeneous internal structures, were divided into five classes characterized by the positions and shapes of high and low electron density areas. Further analysis of the maps in each class by a manifold learning algorithm revealed that the internal structures of Cu2O particles varied in correlation with total electron density while retaining the characteristics within each class. On the basis of the analyses, we proposed a growth mechanism to yield the inhomogeneity in the internal structures of Cu2O particles in surfactant-mediated liquid-phase synthesis.

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生物細胞の低温Ｘ線回折イメージング

中迫雅由，高山裕貴，小林周，山本隆寛，大出真央，岡島公司，苙口友隆，山本雅貴

放射光 31 （ 3 ） 189 - 201 2018年06月

研究論文（学術雑誌）, 共著, 査読有り

Coherent X-ray DIFFRACTION IMAGINg of Cyanidioschyzon merolae

Sekiguchi Y., Kobayashi A., Takayama Y., Oide M., Fukuda A., Yamamoto T., Okajima K., Oroguchi T., Hirakawa T., Inui Y., Matsunaga S., Yamamoto M., Nakasako M.

Cyanidioschyzon merolae: A New Model Eukaryote for Cell and Organelle Biology （Cyanidioschyzon merolae: A New Model Eukaryote for Cell and Organelle Biology）     153 - 173 2018年03月

ISSN  9789811061004

　概要を見る

© Springer Nature Singapore Pte Ltd. 2017. Coherent X-ray diffraction imaging (CXDI) is a lensless imaging technique for visualizing the structures of noncrystalline particles at a resolution of several tens of nanometers. The targets are particles with dimensions in the sub-micrometer to micrometer range. We carried out CXDI experiments at 66 K to investigate the internal structures of a whole frozen-hydrated cell and a chloroplast of Cyanidioschyzon merolae and a cyanobacteria cell by using coherent X-ray light sources, such as synchrotron and X-ray free-electron laser facilities. Owing to the short wavelength of the X-rays used (0.225 nm), the absorption and multiple scattering of X-rays inside the specimens were negligible. Diffraction patterns from each specimen particle adsorbed onto a thin membrane were collected at resolutions better than 50 μm -1 in reciprocal space. Therefore, structures of specimen particles can be illustrated at a resolution higher than 200 nm in real space. The most probable electron density map was retrieved from each diffraction pattern. Theinternal structures of the specimens are described here, particularly the structural correlation of the chloroplast of C. merolae and the cyanobacteria cell. Based on the experimental results, we discuss the feasibility of CXDI in the structural analyses of biological cells and cellular organelles.

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Blue light– excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from arabidopsis

Oide M., Okajima K., Nakagami H., Kato T., Sekiguchi Y., Oroguchi T., Hikima T., Yamamoto M., Nakasako M.

Journal of Biological Chemistry （Journal of Biological Chemistry）  293 （ 3 ） 963 - 972 2018年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00219258

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© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage–sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from Arabidopsis to study their molecular properties in the dark and under BL irradiation. Phosphoryl-ation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2.

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Shot-by-shot characterization of focused X-ray free electron laser pulses

Kobayashi A., Sekiguchi Y., Oroguchi T., Yamamoto M., Nakasako M.

Scientific Reports （Nature publishing）  8 （ 1 ） 831 (1-13) 2018年01月

研究論文（学術雑誌）, 共著, 査読有り

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© 2018 The Author(s). X-ray free electron lasers (XFEL) provide intense and almost coherent X-ray pulses. They are used for various experiments investigating physical and chemical properties in materials and biological science because of their complete coherence, high intensity, and very short pulse width. In XFEL experiments, specimens are irradiated by XFEL pulses focused by mirror optics. The focused pulse is too intense to measure its coherence by placing an X-ray detector on the focal spot. Previously, a method was proposed for evaluating the coherence of focused pulses from the visibility of the diffraction intensity of colloidal particles by the speckle visibility spectroscopy (SVS). However, the visibility cannot be determined exactly because the diffraction intensity is integrated into each finite size detector pixel. Here, we propose a method to evaluate the coherence of each XFEL pulse by using SVS in combination with a theory for exact sampling of the diffraction pattern and a technique of multiplying the diffraction data by a Gaussian masks, which reduces the influence of data missing in small-angle regions due to the presence of a direct beamstop. We also introduce a method for characterizing the shot-by-shot size of each XFEL pulse by analysing the X-ray irradiated area.

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Influences of lone-pair electrons on directionality of hydrogen bonds formed by hydrophilic amino acid side chains in molecular dynamics simulation

Tomotaka Oroguchi and M. Nakasako

Scientific Reports （Nature Publishing）  7   15859 (1-14) 2017年11月

共著, 査読有り

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A protocol for searching most probable phase-retrieved maps in coherent X-ray diffraction imaging by exploiting relationship between convergence of retrieved phase and success of calculation

Yuki Sekiguchi, Saki Hashimoto, Amane Kobayashi, Tomotaka Oroguchi and Masayoshi Nakasako

Journal of Synchrotron Radiation 24   1024 - 1038 2017年09月

研究論文（学術雑誌）, 共著, 査読有り

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Controlled Terahertz Birefringence in Stretched Poly(lactic acid) Films Investigated by Terahertz Time-Domain Spectroscopy and Wide-Angle X‑ray Scattering

Hotsumi Iwasaki, Madoka Nakamura, Nozomu Komatsubara, Makoto Okano, Masayoshi Nakasako, Harumi Sato and Shinichi Watanab

Journal of Physical Chemistry B 121   6951 - 6957 2017年07月

研究論文（学術雑誌）, 共著, 査読有り

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Blue-light-excited LOV2 triggers a rearrangement of the kinase domain to induce phosphorylation activity in Arabidopsis phototropin1

Mao Oide, Koji Okajima, Sachiko Kashojiya, Yuki Takayama, Tomotaka Oroguchi, Takaaki Hikima, Masaki Yamamoto, and Masayoshi Nakasako

Journal of Biological Chemistry 291 （ 38 ） 19975 - 19984 2016年09月

共著, 査読有り

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Common structural features of toxic intermediates from alpha-synuclein and GroES fibrillogenesis detected using cryogenic coherent X-ray diffraction imaging

H. Kameda, S. Usugi, M. Kobayashi, N. Fukui, S. Lee, K. Hongo, T. Mizobata, Y. Sekiguchi, Y. Masaki3, A. Kobayashi, T. Oroguchi, M. Nakasako, Y. Takayama, M. Yamamoto, and Y. Kawata

Journal of Biochemistry (Tokyo) 161   55 - 65 2016年08月

研究論文（学術雑誌）, 共著, 査読有り

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Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using X-ray free electron laser at SACLA

Amane Kobayashi, Yuki Sekiguchi, Tomotaka Oroguchi, Koji Okajima, Asahi Fukuda, Mao Oide, Masaki Yamamoto and Masayoshi Nakasako

Journal of Synchrotron Radiation 23   975 - 989 2016年06月

共著, 査読有り

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Changes in hydration structure are necessary for collective motions of a multi-domain protein

Tomotaka Oroguchi and Masayoshi Nakasako

Scientific Reports 6   26302 (14 pages) 2016年05月

共著, 査読有り

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TAKASAGO-6 apparatus for cryogenic coherent X-ray diffraction imaging of biological non-crystalline particles using X-ray free electron laser at SACLA

Amane Kobayashi, Yuki Sekiguchi, Yuki Takayama, Tomotaka Oroguchi, Keiya Shirahama,　Yasufumi Torizuka, Masahiro Manoda, Masayoshi Nakasako, Masaki Yamamoto

Review of Scientific Instruments 87   053109 (15 pages) 2016年05月

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Classification and assessment of retrieved electron density maps in coherent X-ray diffraction imaging using multivariate analysis

Yuki Sekiguchi, Tomotaka Oroguchi, and Masayoshi Nakasako

Journal of Synchrotron Radiation 23   312 - 323 2015年12月

研究論文（学術雑誌）, 共著, 査読有り

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金コロイド粒子を利用した非結晶生体試料の高分解能・高精度コヒーレントX線回折イメージング

高山裕貴、眞木さおり、苙口友隆、中迫雅由、米倉功治

Isotope News 739 （ 11 ） 9 - 14 2015年11月

研究論文（学術雑誌）, 共著, 査読有り

Classification of projection images of proteins with structural polymorphism by manifold: A simulation study for X-ray free-electron laser diffraction imaging

Takashi Yoshidome, Tomotaka Oroguchi, Masayoshi Nakasako, and Mitsunori Ikeguchi

Physical Review E 92 （ 3 ） 032710 - 13 pages 2015年09月

研究論文（学術雑誌）, 共著, 査読有り

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我が国における生体分子・粒子の構造解析－過去・現在そして未来の展望

中迫雅由、山本雅貴

日本物理学会誌 70 （ 9 ） 702 - 712 2015年09月