Details of the Researcher

PHOTO

Koji Yonekura
Section
Institute of Multidisciplinary Research for Advanced Materials
Job title
Professor
Degree

  • 博士（理学）（東京工業大学）

  • 修士（理学）（東京工業大学）

Awards 5

  1. 栄峰賞

    2025/03　理化学研究所　国産クライオ電顕の開発と電荷・水素原子・化学結合の可視化

  2. 瀬藤賞（日本顕微鏡学会学会賞）

    2024/06　公益社団法人日本顕微鏡学会　クライオ電子顕微鏡による生体分子構造解析技術の開発と応用

  3. 梅峰賞

    2022/03　理化学研究所　Cryo-EMによる生体超分子複合体の構造解析とAI自動測定技術の開発

  4. The Ernst Ruska Prize 2009

    2009/09　The German Society for Electron Microscopy e.V. (DGE)　Contribution Toward Elucidating the Mechanisms of Biological Macromolecular Machines by Cryo-Electron Microscopy

  5. Keck fellow

    2004/02　UCSF

Papers 146

  1. Low-Dose Elemental Mapping of Light Atoms in Liquid-phase Materials Using Cryo-EELS International-journal Peer-reviewed

    Daisuke Unabara, Yohei K. Sato, Tasuku Hamaguchi, Koji Yonekura

    Analytical Chemistry　97　(33)　18055-18063　2025/07/31

    Publisher: American Chemical Society

  2. Versatile Nanoparticle Capsule Formation With Enhanced Encapsulation Efficiency via Solute-Induced Liquid–Liquid Phase Separation International-journal Peer-reviewed

    Takehiro Yachi, Honoka Watanabe, Rumi Niwa, Daisuke Unabara, Tasuku Hamaguchi, Yusuke Yonamine, Koji Yonekura, Kuniharu Ijiro, Hideyuki Mitomo

    Small　2502573　2025/05/07

    Publisher: Wiley-VCH GmbH

  3. Bottom-up design of peptide shapes in water using oligomers of N-methyl-L/D-alanine International-journal Peer-reviewed

    Jumpei Morimoto, Marin Yokomine, Yota Shiratori, Takumi Ueda, Takayuki Nakamuro, Kiyofumi Takaba, Saori Maki-Yonekura, Koji Umezawa, Koichiro Miyanishi, Yasuhiro Fukuda, Takumu Watanabe, Mayuko Suga, Ayumi Inayoshi, Takuya Yoshida, Wataru Mizukami, Koh Takeuchi, Koji Yonekura, Eiichi Nakamura, Shinsuke Sando

    Chemical Science　16　2025/05/02

    Publisher: Royal Society of Chemistry (RSC)

    DOI： 10.1039/d5sc01483b 　

    ISSN： 2041-6520

    eISSN： 2041-6539

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    Conformationally constrained N-methyl-l/d-alanine residues enable the bottom-up design of diverse peptide shapes.

  4. クライオTEMと3次元電子回折法の新展開 Invited

    米倉 功治

    60　(5月号)　324-328　2025/05

    Publisher: 日本セラミックス協会

  5. 3D Electron Diffraction Structure of an Organic Semiconductor Reveals Conformational Polymorphism International-journal Peer-reviewed

    Hirofumi Kurokawa, Saori Maki-Yonekura, Kiyofumi Takaba, Toshiki Higashino, Satoru Inoue, atsuo Hasegawa, Koji Yonekura

    Journal of the American Chemical Society　2025/02/06

    Publisher: ACS Publications

    DOI： 10.26434/chemrxiv-2024-pslhg 　

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    Crystal and conformational polymorphisms play a crucial role in the physical and chemical properties of materials, impacting their stability, solubility, and bioavailability, which are essential for various applications in pharmaceuticals, materials science, and chemistry. Despite their significance, the structural analysis of these polymorphisms, particularly conformational polymorphisms, remains challenging due to the limited methodology that provides sufficient resolution for microcrystalline variants of polymorphs. Three-dimensional electron diffraction (3D ED) is an emerging technique with significant potential for elucidating the microcrystal structures of functional organic molecules, pharmaceuticals, and biomolecules. Despite this potential, there are limited instances of 3D ED structures for small molecules exhibiting the lowest crystallographic symmetry with a preferred orientation, and possibly conformational variations of constituent molecules. A novel organic semi-conductor, Ph-anti-benzothieno[5,6-b]benzothieno[3,2-b]thiophene-C10 (antiC10) is one of such examples. We have successfully determined the 3D ED structure of this challenging molecule for the first time. The antiC10 crystal exhibited the lowest symmetry (space group P1), and the preferred orientations against the grid resulted in a missing cone. These challenges were surmounted by employing a sequential molecular replacement approach with an ab initio generated search model. The resulting octameric antiC10 structure reveals a two-monolayer architecture and an antiparallel alkyl-interdigitated herringbone configuration, in contrast to the all-parallel associations observed in its previously reported isomer. Concurrently, the alkyl chains intricately interdigitated with each other, positioned between the adjacent π-core strata. Detailed analysis has elucidated the conformational polymorphism in herringbone packing between the two monolayers, as well as in intramolecular conformations among monomers. The structure with conformational polymorphism is presumably in a metastable intermediate state stabilized by twinning. These findings may provide critical insights into the crystallization mechanisms and rational design of organic semiconductors. This research demonstrates that advancements in 3D ED technology and sequential phasing methodologies have enabled the study of previously unreachable structures.

  6. High throughput protein serial crystallography using a grease matrix and a large-area support film

    Michihiro Sugahara, Saori Maki-Yonekura, Ichiro Inoue, Kiyofumi Takaba, Shun Narai, Hisashi Naitow, Jungmin Kang, Kensuke Tono, Keiji Numata, Tetsuya Ishikawa, Makina Yabashi, Koji Yonekura

    bioRxiv　2025/01/31

    Publisher: Cold Spring Harbor Laboratory

    DOI： 10.1101/2025.01.31.635837 　

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    Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free-electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

  7. Streamlined modular synthesis of saframycin substructure via copper-catalyzed three-component assembly and gold-promoted 6-endo cyclization Peer-reviewed

    Asahi Kanno, Ryo Tanifuji, Satoshi Yoshida, Sota Sato, Saori Maki-Yonekura, Kiyofumi Takaba, Jungmin Kang, Kensuke Tono, Koji Yonekura, Hiroki Oguri

    The Beilstein Journal of Organic Chemistry　221　226-233　2025/01/28

    Publisher: Beilstein Institute for the Advancement of Chemical Sciences

    DOI： 10.3762/bjoc.21.14 　

  8. Exploration and development of molecule-based printed electronics materials: an integrated approach using experimental, computational, and data sciences

    Tatsuo Hasegawa, Satoru Inoue, Seiji Tsuzuki, Sachio Horiuchi, Hiroyuki Matsui, Tomoharu Okada, Reiji Kumai, Koji Yonekura, Saori Maki-Yonekura

    Science and Technology of Advanced Materials　25　(1)　2418282　2024/12/06

    Publisher: Informa UK Limited

    DOI： 10.1080/14686996.2024.2418282 　

    ISSN： 1468-6996

    eISSN： 1878-5514

  9. A graph-based molecular structure identification method via feature extraction for three-dimensional electron diffraction Data

    Yusuke Fukasawa, Kazuhiko Komatsu, Masayuki Sato, Saori Maki-Yonekura, Hirofumi Kurokawa, Koji Yonekura, Hiroaki Kobayashi

    2024 Twelfth International Symposium on Computing and Networking Workshops (CANDARW)　325-329　2024/11/26

    Publisher: IEEE

    DOI： 10.1109/candarw64572.2024.00060 　

  10. Emergence of mixing-induced polymorphic phase with higher mobility in alkylated layered organic semiconductors

    Kiyoshi Nikaido, Satoru Inoue, Seiji Tsuzuki, Reiji Kumai, Hiroyuki Matsui, Kiyofumi Takaba, Saori Maki-Yonekura, Koji Yonekura, Tatsuo Hasegawa

    Physical Review Materials　8　115601　2024/11/01

    Publisher: American Physical Society (APS)

    DOI： 10.1103/physrevmaterials.8.115601 　

    eISSN： 2475-9953

  11. Artificial Intelligence-Driven Data Acquisition for Single-Particle Cryo-EM and Three-Dimensional Electron Diffraction Invited Peer-reviewed

    Koji Yonekura

    Cryo-Electron Microscopy in Structural Biology　2024/10/17

    Publisher: CRC Press

    DOI： 10.1201/9781003326106 　

  12. Structure of endothelin ETB receptor–Gi complex in a conformation stabilized by unique NPxxL motif

    Kazutoshi Tani, Saori Maki-Yonekura, Ryo Kanno, Tatsuki Negami, Tasuku Hamaguchi, Malgorzata Hall, Akira Mizoguchi, Bruno M. Humbel, Tohru Terada, Koji Yonekura, Tomoko Doi

    Communications Biology　7　1303　2024/10/16

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s42003-024-06905-z 　

    eISSN： 2399-3642

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    Abstract Endothelin type B receptor (ETBR) plays a crucial role in regulating blood pressure and humoral homeostasis, making it an important therapeutic target for related diseases. ETBR activation by the endogenous peptide hormones endothelin (ET)−1–3 stimulates several signaling pathways, including Gs, Gi/o, Gq/11, G12/13, and β-arrestin. Although the conserved NPxxY motif in transmembrane helix 7 (TM7) is important during GPCR activation, ETBR possesses the lesser known NPxxL motif. In this study, we present the cryo-EM structure of the ETBR–Gi complex, complemented by MD simulations and functional studies. These investigations reveal an unusual movement of TM7 to the intracellular side during ETBR activation and the essential roles of the diverse NPxxL motif in stabilizing the active conformation of ETBR and organizing the assembly of the binding pocket for the α5 helix of Gi protein. These findings enhance our understanding of the interactions between GPCRs and G proteins, thereby advancing the development of therapeutic strategies.

  13. XFELと電子顕微鏡の相補利用による微小結晶構造解析 Invited

    高場圭章, 眞木さおり, 米倉功治

    放射光　3　(5)　297-303　2024/09/30

  14. クライオ電子顕微鏡で電荷・化学結合を可視化 Invited

    眞木 さおり, 川上 恵典, 米倉 功治

    Isotope News　4-8　2024/06

    Publisher: 日本アイソトープ協会

  15. The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs

    Koji Kato, Tasuku Hamaguchi, Minoru Kumazawa, Yoshiki Nakajima, Kentaro Ifuku, Shunsuke Hirooka, Yuu Hirose, Shin-ya Miyagishima, Takehiro Suzuki, Keisuke Kawakami, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen, Ryo Nagao

    Proceedings of the National Academy of Sciences　121　(11)　e2319658121　2024/03/05

    Publisher: Proceedings of the National Academy of Sciences

    DOI： 10.1073/pnas.2319658121 　

    ISSN： 0027-8424

    eISSN： 1091-6490

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    Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae , whereas no orthologous relationships were found between C. caldarium Lhcr1–3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

  16. Comprehensive Application of XFEL Microcrystallography for Challenging Targets in Various Organic Compounds

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Yasuhiro Fukuda, Yota Shiratori, Yiying Peng, Jumpei Morimoto, Satoru Inoue, Toshiki Higashino, Shinsuke Sando, Tatsuo Hasegawa, Makina Yabashi, Koji Yonekura

    Journal of the American Chemical Society　146　(9)　5872-5882　2024/02/28

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/jacs.3c11523 　

    ISSN： 0002-7863

    eISSN： 1520-5126

  17. Reinvestigation on primary processes of PSII-dimer from Thermosynechococcus vulcanus by femtosecond pump-probe spectroscopy

    Daisuke Kosumi, Miki Bandou-Uotani, Shunya Kato, Keisuke Kawakami, Koji Yonekura, Nobuo Kamiya

    Photosynthesis Research　159　79-91　2024/02/24

    Publisher: Springer

    DOI： 10.1007/s11120-024-01076-8 　

    ISSN： 0166-8595

    eISSN： 1573-5079

  18. XFEL and electron microcrystallography of small organic compounds -measuring charges and chemical bonds of hydrogen atoms using the two beam sources-

    YONEKURA Koji, TAKABA Kiyofumi, MAKI-YONEKURA Saori

    SPring-8/SACLA Information　29　(1)　14-18　2024/02/15

  19. Effects of Thiophene-Fused Isomer on High-Layered Crystallinity in π-Extended and Alkylated Organic Semiconductors

    Toshiki Higashino, Satoru Inoue, Shunto Arai, Seiji Tsuzuki, Hiroyuki Matsui, Reiji Kumai, Kiyofumi Takaba, Saori Maki-Yonekura, Hirofumi Kurokawa, Ichiro Inoue, Kensuke Tono, Koji Yonekura, Tatsuo Hasegawa

    Chemistry of Materials　36　(2)　848-859　2024/01/05

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/acs.chemmater.3c02500 　

    ISSN： 0897-4756

    eISSN： 1520-5002

  20. Applications and limitations of electron 3D crystallography International-journal Invited

    Koji Yonekura, Saori Maki-Yonekura, Kiyofumi Takaba

    Structure　31　(11)　1328-1334　2023/11/02

    Publisher: Elsevier BV

    DOI： 10.1016/j.str.2023.09.007 　

    ISSN： 0969-2126

  21. Structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs

    Koji Kato, Tasuku Hamaguchi, Minoru Kumazawa, Yoshiki Nakajima, Kentaro Ifuku, Shunsuke Hirooka, Yuu Hirose, Shin-ya Miyagishima, Takehiro Suzuki, Keisuke Kawakami, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen, Ryo Nagao

    BioRxiv　2023/10/27

    Publisher: Cold Spring Harbor Laboratory

    DOI： 10.1101/2023.10.25.563911 　

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    Abstract Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and their structural variety in photosystem I-LHC (PSI-LHCI) supercomplexes has been shown. However, structural and evolutionary correlations of red-lineage LHCs are unknown. Here we determined a 1.92-Å resolution cryo-electron microscopic structure of a PSI-LHCI supercomplex isolated from the red algaCyanidium caldariumRK-1 (NIES-2137) which is an important taxon in the Cyanidiophyceae, and subsequently investigated these correlations through structural comparisons and phylogenetic analysis. The PSI-LHCI structure shows five LHCI subunits together with a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in red-lineage algae showed clear orthology of LHCs betweenC. caldariumandCyanidioschyzon merolae, whereas no orthologous relationships were found betweenC. caldariumLhcr1–3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

  22. AI制御によるクライオEMの自動測定システム Invited

    米倉 功治

    月刊「細胞」　55　(13)　4-7　2023/10/20

    Publisher: 北隆館

  23. Comprehensive application of XFEL micro crystallography for novel organic compounds

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Yasuhiro Fukuda, Yota Shiratori, Yiying Peng, Jumpei Morimoto, Satoru Inoue, Toshiki Higashino, Shinsuke Sando, Tatsuo Hasegawa, Makina Yabashi, Koji Yonekura

    ChemRxiv　2023/10/05

    Publisher: American Chemical Society (ACS)

    DOI： 10.26434/chemrxiv-2023-69p99 　

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    There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to tiny crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate, coupled with rotation, enables highly efficient data collection. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferred-oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data, regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies on small crystals of organic compounds.

  24. Synthesis of (±)-zeapyranolactone, a noncanonical strigolactone isolated from maize, and determination of its overall relative configuration International-journal Peer-reviewed

    Chinatsu Ono-Ogasawara, Shunya Yamamoto, Yusuke Ogura, Hironori Okamura, Kiyofumi Takaba, Satoshi Yoshida, Sota Sato, Koji Yonekura, Hirosato Takikawa

    Tetrahedron Letters　127　(1)　1-4　2023/09

    Publisher: Elsevier

    DOI： 10.1016/j.tetlet.2023.154695 　

  25. クライオ電顕で見るタンパク質の化学特性――水素原子，電荷，化学結合も可視化する新たなステージへ Invited

    米倉 功治, 眞木さおり, 川上恵典

    化学　78　(9)　39-43　2023/08/18

    Publisher: 株式会社 化学同人

  26. Enantioselectivity of discretized helical supramolecule consisting of achiral cobalt phthalocyanines via chiral-induced spin selectivity effect

    Hiroki Aizawa, Takuro Sato, Saori Maki-Yonekura, Koji Yonekura, Kiyofumi Takaba, Tasuku Hamaguchi, Taketoshi Minato, Hiroshi M. Yamamoto

    Nature Communications　14　(1)　4530　2023/07/28

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s41467-023-40133-z 　

    eISSN： 2041-1723

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    Abstract Enantioselectivity of helical aggregation is conventionally directed either by its homochiral ingredients or by introduction of chiral catalysis. The fundamental question, then, is whether helical aggregation that consists only of achiral components can obtain enantioselectivity in the absence of chiral catalysis. Here, by exploiting enantiospecific interaction due to chiral-induced spin selectivity (CISS) that has been known to work to enantio-separate a racemic mixture of chiral molecules, we demonstrate the enantioselectivity in the assembly of mesoscale helical supramolecules consisting of achiral cobalt phthalocyanines. The helical nature in our supramolecules is revealed to be mesoscopically incorporated by dislocation-induced discretized twists, unlike the case of chiral molecules whose chirality are determined microscopically by chemical bond. The relevance of CISS effect in the discretized helical supramolecules is further confirmed by the appearance of spin-polarized current through the system. These observations mean that the application of CISS-based enantioselectivity is no longer limited to systems with microscopic chirality but is expanded to the one with mesoscopic chirality.

  27. Measurement of charges and chemical bonding in a cryo-EM structure Peer-reviewed

    Saori Maki-Yonekura, Keisuke Kawakami, Kiyofumi Takaba, Tasuku Hamaguchi, Koji Yonekura

    Communications Chemistry　6　(1)　98　2023/05/31

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s42004-023-00900-x 　

    eISSN： 2399-3669

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    Abstract Hydrogen bonding, bond polarity, and charges in protein molecules play critical roles in the stabilization of protein structures, as well as affecting their functions such as enzymatic catalysis, electron transfer, and ligand binding. These effects can potentially be measured in Coulomb potentials using cryogenic electron microscopy (cryo-EM). We here present charges and bond properties of hydrogen in a sub-1.2 Å resolution structure of a protein complex, apoferritin, by single-particle cryo-EM. A weighted difference map reveals positive densities for most hydrogen atoms in the core region of the complex, while negative densities around acidic amino-acid side chains are likely related to negative charges. The former positive densities identify the amino- and oxo-termini of asparagine and glutamine side chains. The latter observations were verified by spatial-resolution selection and a dose-dependent frame series. The average position of the hydrogen densities depends on the parent bonded-atom type, and this is validated by the estimated level of the standard uncertainties in the bond lengths.

  28. Bottom-up design of peptide nanoshapes in water using oligomers of N-methyl-L/D-alanine

    Jumpei Morimoto, Yota Shiratori, Marin Yokomine, Takumi Ueda, Takayuki Nakamuro, Kiyofumi Takaba, Saori Maki-Yonekura, Koji Umezawa, Koichiro Miyanishi, Yasuhiro Fukuda, Takumu Watanabe, Wataru Mizukami, Koh Takeuchi, Koji Yonekura, Eiichi Nakamura, Shinsuke Sando

    ChemRxiv　2023/05/16

    Publisher: American Chemical Society (ACS)

    DOI： 10.26434/chemrxiv-2023-c4mw6 　

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    De novo design of peptide nanoshapes is of great interest in biomolecular science since the local peptide nanoshapes formed by a short peptide chain in the proteins are often key to the biological activities. Here, we show that the de novo design of peptide nanoshapes with sub-nanometer conformational control can be realized using peptides consisting of N-methyl-L-alanine and N-methyl-D-alanine residues as studied by NMR, X-ray and XFEL crystallographic and computational analyses as well as by direct imaging of the dynamics of the peptide’s nanoshape using cinematographic electron microscopic technique. The conformation of N-methyl-L/D-alanine residue is largely fixed because of the restricted bond rotation, and hence can serve as a scaffold on which we can build a peptide into a designed nanoshape. The local shape control by per-residue conformational restriction by torsional strains starkly contrasts with the global shape stabilization of proteins based on many remote interactions. The oligomers allow the bottom-up design of diverse peptide nanoshapes with a small number of amino acid residues and would offer unique opportunities to realize the de novo design of biofunctional molecules, such as catalysts and drugs.

  29. Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography Peer-reviewed

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Tasuku Hamaguchi, Keisuke Kawakami, Hisashi Naitow, Tetsuya Ishikawa, Makina Yabashi, Koji Yonekura

    Nature Chemistry　15　491-497　2023/03/20

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s41557-023-01162-9 　

    ISSN： 1755-4330

    eISSN： 1755-4349

  30. Unnaturally Distorted Hexagonal Protein Ring Alternatingly Reorganized from Two Distinct Chemically Modified Proteins Peer-reviewed

    Tomoki Himiyama, Tasuku Hamaguchi, Koji Yonekura, Tsutomu Nakamura

    Bioconjugate Chemistry　34　(4)　764-771　2023/03/08

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/acs.bioconjchem.3c00057 　

    ISSN： 1043-1802

    eISSN： 1520-4812

  31. フィコビリソームが太陽光エネルギーを吸収する仕組み

    川上恵典, 米倉功治

    バイオサイエンスとインダストリー　81　(2)　113-115　2023/03

  32. Ultrafast energy transfer and charge separation dynamics in PSII-dimer from Thermosynechococcus vulcanus

    Daisuke Kosumi, Miki Bandou-Uotani, Shunya Kato, Keisuke Kawakami, Koji Yonekura, Nobuo Kamiya

    Research Square　2023/02/27

    Publisher: Research Square Platform LLC

    DOI： 10.21203/rs.3.rs-2613905/v1 　

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    Abstract Cyanobacterial photosynthetic apparatus efficiently capture sunlight, and the energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. PSII is a unique membrane protein complex that photo-catalyzes oxidation of water and majorly contains photosynthetic pigments of chlorophyll a and carotenoids. In the present study, the ultrafast energy transfer and charge separation dynamics of PSII from a thermophilic cyanobacteria Thermosynechococcus vulcanus have been investigated by femtosecond pump-probe spectroscopic measurements. The results demonstrated that the pheophytin anion PheoD1− generates with 43.0 ps at 140 K (14.8 ps at 296 K) and the secondary electron transfer to quinone: PheoD1−QA →PheoD1QA− occurs with 650 ps at 296 K upon excitation into the Qy band of chlorophyll a at 670 nm. On the other hand, an inefficient β-carotene → chlorophyll a energy transfer (33%) occurred after excitation to the S2 state of β-carotene at 500 nm. Instead, the carotenoid triplet state appeared in an ultrafast timescale after excitation at 500 nm.

  33. Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120

    Ryo Nagao, Koji Kato, Tasuku Hamaguchi, Yoshifumi Ueno, Naoki Tsuboshita, Shota Shimizu, Miyu Furutani, Shigeki Ehira, Yoshiki Nakajima, Keisuke Kawakami, Takehiro Suzuki, Naoshi Dohmae, Seiji Akimoto, Koji Yonekura, Jian-Ren Shen

    Nature Communications　14　(1)　920　2023/02/17

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s41467-023-36504-1 　

    eISSN： 2041-1723

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    Abstract Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits were identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 occupies the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a PSI monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.

  34. AIクライオEMデータ測定 Invited

    米倉 功治

    クライオ電子顕微鏡ハンドブック　293-302　2023/01

  35. 電子線三次元結晶構造解析/3D ED/マイクロEDの解析事例 Invited

    眞木 さおり, 高場 圭章, 米倉功治

    クライオ電子顕微鏡ハンドブック　257-259　2023/01

  36. 電子線三次元結晶構造解析/3D ED/マイクロEDの原理、他法との比較、展望 Invited

    米倉功治, 眞木さおり, 高場 圭章

    クライオ電子顕微鏡ハンドブック　35-44　2023/01

  37. 電子線三次元結晶構造解析/3D ED/マイクロED の解析例と展望 Invited

    米倉功治, 眞木さおり, 高場 圭章

    顕微鏡　57　118-123　2022/12

  38. ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures International-journal Peer-reviewed

    Naotaka Sekiyama, Kiyofumi Takaba, Saori Maki-Yonekura, Ken-ichi Akagi, Yasuko Ohtani, Kayo Imamura, Tsuyoshi Terakawa, Keitaro Yamashita, Daigo Inaoka, Koji Yonekura, Takashi S. Kodama, Hidehito Tochio

    Proceedings of the National Academy of Sciences　119　(38)　e2122523119　2022/09/20

    Publisher: Proceedings of the National Academy of Sciences

    DOI： 10.1073/pnas.2122523119 　

    ISSN： 0027-8424

    eISSN： 1091-6490

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    T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.

  39. Chl dを持つAcaryochloris marinaの新型光化学系Ⅰ複合体の構造

    川上 恵典, 伊藤 繁, 菓子野 康浩

    光合成研究　32　(2)　95-106　2022/08

    Publisher: 日本光合成研究会

  40. Core and rod structures of a thermophilic cyanobacterial light-harvesting phycobilisome International-journal Peer-reviewed

    Keisuke Kawakami, Tasuku Hamaguchi, Yuu Hirose, Daisuke Kosumi, Makoto Miyata, Nobuo Kamiya, Koji Yonekura

    Nature Communications　13　(1)　3389　2022/06

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s41467-022-30962-9 　

    eISSN： 2041-1723

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    Abstract Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.

  41. 電子線と放射光での結晶化学

    高場圭章

    日本結晶学会誌　64　(2)　115-116　2022/05/31

  42. Theoretical Model of the Far-Red-Light-Adapted Photosystem I Reaction Center of Cyanobacterium Acaryochloris marina Using Chlorophyll d and the Effect of Chlorophyll Exchange

    Akihiro Kimura, Hirotaka Kitoh-Nishioka, Toshimichi Aota, Tasuku Hamaguchi, Koji Yonekura, Keisuke Kawakami, Kyoko Shinzawa-Itoh, Natsuko Inoue-Kashino, Kentaro Ifuku, Eiki Yamashita, Yasuhiro Kashino, Shigeru Itoh

    The Journal of Physical Chemistry B　126　(22)　4009-4021　2022/05/26

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/acs.jpcb.2c00869 　

    ISSN： 1520-6106

    eISSN： 1520-5207

  43. Structural basis for the absence of low-energy chlorophylls in a photosystem I trimer from Gloeobacter violaceus International-journal Peer-reviewed

    Koji Kato, Tasuku Hamaguchi, Ryo Nagao, Keisuke Kawakami, Yoshifumi Ueno, Takehiro Suzuki, Hiroko Uchida, Akio Murakami, Yoshiki Nakajima, Makio Yokono, Seiji Akimoto, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen

    eLife　11　2022/04/11

    Publisher: eLife Sciences Publications, Ltd

    DOI： 10.7554/elife.73990 　

    eISSN： 2050-084X

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    Photosystem I (PSI) is a multi-subunit pigment-protein complex that functions in light-harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation in photosynthetic organisms. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the sites of low-energy Chls are still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primordial cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure shows the absence of some subunits commonly found in other cyanobacteria, confirming the primordial nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 is missing in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. These findings provide insights into not only the identity of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls in oxyphototrophs.

  44. Emerging Disordered Layered-Herringbone Phase in Organic Semiconductors Unveiled by Electron Crystallography Peer-reviewed

    Satoru Inoue, Kiyoshi Nikaido, Toshiki Higashino, Shunto Arai, Mutsuo Tanaka, Reiji Kumai, Seiji Tsuzuki, Sachio Horiuchi, Haruki Sugiyama, Yasutomo Segawa, Kiyofumi Takaba, Saori Maki-Yonekura, Koji Yonekura, Tatsuo Hasegawa

    Chemistry of Materials　34　(1)　72-83　2022/01/11

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/acs.chemmater.1c02793 　

    ISSN： 0897-4756

    eISSN： 1520-5002

  45. Hydrogen properties and charges in a sub-1.2 Å resolution cryo-EM structure revealed by a cold field emission beam

    Saori Maki-Yonekura, Keisuke Kawakami, Tasuku Hamaguchi, Kiyofumi Takaba, Koji Yonekura

    2021/12/22

    Publisher: Cold Spring Harbor Laboratory

    DOI： 10.1101/2021.12.21.473430 　

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    Abstract The cold field emission (CFE) beam produces the less-attenuated contrast transfer function of electron microscopy, thereby enhancing high-resolution signals and this particularly benefits higher-resolution single particle cryogenic electron microscopy. Here, we present a sub-1.2 Å resolution structure of a standard protein sample, apoferritin. Image data were collected with the CFE beam in a high-throughput scheme while minimizing beam tilt deviations from the coma-free axis. A difference map reveals positive densities for most hydrogen atoms in the core region of the protein complex including those in water molecules, while negative densities around acidic amino-acid side chains likely represent negative charges. The position of the hydrogen densities depends on parent bonded-atom type, which is validated by an estimated level of coordinate errors.

  46. Machine learning-based real-time object locator/evaluator for cryo-EM data collection Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Hisashi Naitow, Tasuku Hamaguchi, Kiyofumi Takaba

    Communications Biology　4　(1)　1044　2021/12

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s42003-021-02577-1 　

    eISSN： 2399-3642

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    <title>Abstract</title>In cryo-electron microscopy (cryo-EM) data collection, locating a target object is error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation shows its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and in locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection. The proposed approach will advance high-throughput and accurate data collection of images and diffraction patterns with minimal human operation.

  47. Hydrogen properties in an organic molecule revealed by XFEL and electron crystallography

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Tasuku Hamaguchi, Keisuke Kawakami, Hisahi Naitow, Tetsuya Ishikawa, Makina Yabashi, Koji Yonekura

    2021/11/09

    Publisher: American Chemical Society (ACS)

    DOI： 10.26434/chemrxiv-2021-jvbfl 　

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    Structure analysis of small crystals is important in synthetic organic chemistry, pharmaceutical and material sciences, and related areas, as the conformation of these molecules may differ in large and small crystals, thus affecting the interpretation of their functional properties and drug efficacy. From small crystals, X-ray and electron beams could furnish electron densities and Coulomb potentials of target molecules, respectively. The two beams provide distinctly different information, and this potential has not been fully explored. Here we present the detailed structure of an organic molecule, rhodamine-6g by X-ray free-electron laser (XFEL) and electron crystallography from the same sample batch of microcrystals. This is the first organic molecular structure determined using XFEL at subatomic resolution. Direct comparison between the electron-density and the Coulomb-potential maps together with theoretical models based on Poisson’s equation shows that the position of hydrogen atoms depends on bond type and charge distribution. The combined approach could lead to better insights into their chemical and/or binding properties for a broad range of organic molecules.

  48. Structural basis for the absence of low-energy chlorophylls responsible for photoprotection from a primitive cyanobacterial PSI

    Koji Kato, Tasuku Hamaguchi, Ryo Nagao, Keisuke Kawakami, Yoshifumi Ueno, Takehiro Suzuki, Hiroko Uchida, Akio Murakami, Yoshiki Nakajima, Makio Yokono, Seiji Akimoto, Naoshi Dohmae, Koji Yonekura, Jian-Ren Shen

    2021/10/01

    Publisher: Cold Spring Harbor Laboratory

    DOI： 10.1101/2021.09.29.462462 　

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    Abstract Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

  49. Electron 3D Crystallography/3D ED/MicroED Invited Peer-reviewed

    Koji YONEKURA

    Nihon Kessho Gakkaishi　63　(3)　189-196　2021/08/31

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.63.189 　

    ISSN： 0369-4585

    eISSN： 1884-5576

  50. Corrigendum: Protein and Organic-Molecular Crystallography with 300kV Electrons on a Direct Electron Detector (Frontiers in Molecular Biosciences, (2021), 7, (612226), 10.3389/fmolb.2020.612226)

    Kiyofumi Takaba, Saori Maki-Yonekura, Satoru Inoue, Tatsuo Hasegawa, Koji Yonekura

    Frontiers in Molecular Biosciences　8　2021/08/18

    Publisher: Frontiers Media S.A.

    DOI： 10.3389/fmolb.2021.749448 　

    ISSN： 2296-889X

  51. Complementary use of high-resolution and high-precision cryo-ED and EM Invited

    Koji Yonekura, Saori Maki-Yonekura, Kiyofumi Takaba, Tasuku Hamaguchi, Hisashi Naitow, Keisuke Kawakami

    Microscopy and Microanalysis　27　(S1)　204-206　2021/08/01

    Publisher: Cambridge University Press (CUP)

    DOI： 10.1017/s143192762100132x 　

    ISSN： 1431-9276

    eISSN： 1435-8115

  52. Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer Peer-reviewed

    Huaxin Yu, Tasuku Hamaguchi, Yoshiki Nakajima, Koji Kato, Keisuke Kawakami, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

    Biochimica et Biophysica Acta (BBA) - Bioenergetics　148471-148471　2021/07

    Publisher: Elsevier BV

    DOI： 10.1016/j.bbabio.2021.148471 　

    ISSN： 0005-2728

  53. Structural implications for a phycobilisome complex from the thermophilic cyanobacterium Thermosynechococcus vulcanus Peer-reviewed

    Keisuke Kawakami, Ryo Nagao, Yuhei O. Tahara, Tasuku Hamaguchi, Takehiro Suzuki, Naoshi Dohmae, Daisuke Kosumi, Jian-Ren Shen, Makoto Miyata, Koji Yonekura, Nobuo Kamiya

    Biochimica et Biophysica Acta (BBA) - Bioenergetics　1862　(9)　148458-148458　2021/05

    Publisher: Elsevier BV

    DOI： 10.1016/j.bbabio.2021.148458 　

    ISSN： 0005-2728

  54. Structural insights into the targeting specificity of ubiquitin ligase for S.cerevisiae isocitrate lyase but not C.albicans isocitrate lyase International-journal Peer-reviewed

    Keito Hiragi, Kazuya Nishio, Shu Moriyama, Tasuku Hamaguchi, Akira Mizoguchi, Koji Yonekura, Kazutoshi Tani, Tsunehiro Mizushima

    Journal of Structural Biology　213　(3)　107748-107748　2021/05

    Publisher: Elsevier BV

    DOI： 10.1016/j.jsb.2021.107748 　

    ISSN： 1047-8477

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    In Saccharomyces cerevisiae, the glyoxylate cycle is controlled through the posttranslational regulation of its component enzymes, such as isocitrate lyase (ICL), which catalyzes the first unique step of the cycle. The ICL of S.cerevisiae (ScIcl1) is tagged for proteasomal degradation through ubiquitination by a multisubunit ubiquitin ligase (the glucose-induced degradation-deficient (GID) complex), whereas that of the pathogenic yeast Candida albicans (CaIcl1) escapes this process. However, the reason for the ubiquitin targeting specificity of the GID complex for ScIcl1 and not for CaIcl1 is unclear. To gain some insight into this, in this study, the crystal structures of apo ScIcl1 and CaIcl1 in complex with formate and the cryogenic electron microscopy structure of apo CaIcl1 were determined at a resolution of 2.3, 2.7, and 2.6 Å, respectively. A comparison of the various structures suggests that the orientation of N-terminal helix α1 in S.cerevisiae is likely key to repositioning of ubiquitination sites and contributes to the distinction found in C. albicans ubiquitin evasion mechanism. This finding gives us a better understanding of the molecular mechanism of ubiquitin-dependent ScIcl1 degradation and could serve as a theoretical basis for the research and development of anti-C. albicans drugs based on the concept of CaIcl1 ubiquitination.

  55. Structure of the far-red light utilizing photosystem I of Acaryochloris marina Peer-reviewed

    Tasuku Hamaguchi, Keisuke Kawakami, Kyoko Shinzawa-Itoh, Natsuko Inoue-Kashino, Shigeru Itoh, Kentaro Ifuku, Eiki Yamashita, Kou Maeda, Koji Yonekura, Yasuhiro Kashino

    Nature Communications　12　(1)　2333　2021/04

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s41467-021-22502-8 　

    eISSN： 2041-1723

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    <title>Abstract</title><italic>Acaryochloris marina</italic> is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of <italic>A. marina</italic> photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll <italic>d</italic> and its epimer chlorophyll <italic>d</italic>′. The primary electron acceptor is pheophytin <italic>a</italic>, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.

  56. Double-Helix Supramolecular Nanofibers Assembled from Negatively Curved Nanographenes Peer-reviewed

    Kenta Kato, Kiyofumi Takaba, Saori Maki-Yonekura, Nobuhiko Mitoma, Yusuke Nakanishi, Taishi Nishihara, Taito Hatakeyama, Takuma Kawada, Yuh Hijikata, Jenny Pirillo, Lawrence T. Scott, Koji Yonekura, Yasutomo Segawa, Kenichiro Itami

    Journal of the American Chemical Society　143　(14)　5465-5469　2021/03/24

    Publisher: American Chemical Society (ACS)

    DOI： 10.1021/jacs.1c00863 　

    ISSN： 0002-7863

    eISSN： 1520-5126

  57. Advances in cryo-EM and ED with a cold-field emission beam and energy filtration —Refinements of the CRYO ARM 300 system in RIKEN SPring-8 center— Peer-reviewed

    Saori Maki-Yonekura, Tasuku Hamaguchi, Hisashi Naitow, Kiyofumi Takaba, Koji Yonekura

    Microscopy　70　(2)　232-240　2021/03/24

    Publisher: Oxford University Press (OUP)

    DOI： 10.1093/jmicro/dfaa052 　

    ISSN： 2050-5698

    eISSN： 2050-5701

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    <title>Abstract</title> We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope—the first machine of this model—and a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly coherent 300-kV electron beam to a sample from a cold-field emission gun and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress and future plan for running such cryo-EM machines in RIKEN SPring-8 Center.

  58. High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams Peer-reviewed

    Koji Kato, Naoyuki Miyazaki, Tasuku Hamaguchi, Yoshiki Nakajima, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

    Communications Biology　4　(1)　2021/03

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/s42003-021-01919-3 　

    eISSN： 2399-3642

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    <title>Abstract</title>Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

  59. Protein and Organic-Molecular Crystallography With 300kV Electrons on a Direct Electron Detector Invited Peer-reviewed

    Kiyofumi Takaba, Saori Maki-Yonekura, Satoru Inoue, Tatsuo Hasegawa, Koji Yonekura

    Frontiers in Molecular Biosciences　7　2021/01/06

    Publisher: Frontiers Media SA

    DOI： 10.3389/fmolb.2020.612226 　

    eISSN： 2296-889X

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    Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera.

  60. Cryo-EM and ED with a Cold-Field Emission Beam and Energy Filtration Invited Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Tasuku Hamaguchi, Hisashi Naitow, Kiyofumi Takaba, Keisuke Kawakami

    Applications of Microscopy in Materials and Life Sciences　233-241　2021

    Publisher: Springer Singapore

    DOI： 10.1007/978-981-16-2982-2_23 　

    ISSN： 2662-3161

    eISSN： 2662-317X

  61. High-resolution cryo-EM structure of photosystem II: Effects of electron beam damage

    Koji Kato, Naoyuki Miyazaki, Tasuku Hamaguchi, Yoshiki Nakajima, Fusamichi Akita, Koji Yonekura, Jian-Ren Shen

    2020/10/19

    Publisher: Cold Spring Harbor Laboratory

    DOI： 10.1101/2020.10.18.344648 　

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    Abstract Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

  62. Apple latent spherical virus structure with stable capsid frame supports quasi-stable protrusions expediting genome release. International-journal Peer-reviewed

    Hisashi Naitow, Tasuku Hamaguchi, Saori Maki-Yonekura, Masamichi Isogai, Nobuyuki Yoshikawa, Koji Yonekura

    Communications biology　3　(1)　488-488　2020/09/04

    DOI： 10.1038/s42003-020-01217-4 　

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    Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of the survival of these viruses depends on their capsid being stable enough to harbour the viral genome and yet malleable enough to allow its release. However, molecular mechanisms remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with a cold-field emission beam. The cryo-EM map reveals a unique structure composed of three capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits. The structures and observations suggest that genome release occurs through occasional opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame of the other subunits.

  63. Erratum to "A new cryo-EM system for single particle analysis" [J. Struct. Biol. 207 (1) (2019) 40-48]. International-journal Peer-reviewed

    Tasuku Hamaguchi, Saori Maki-Yonekura, Hisashi Naitow, Yoshinori Matsuura, Tetsuya Ishikawa, Koji Yonekura

    Journal of structural biology　211　(3)　107558-107558　2020/09/01

    DOI： 10.1016/j.jsb.2020.107558 　

  64. Collecting large datasets of rotational electron diffraction with ParallEM and SerialEM. International-journal Peer-reviewed

    Kiyofumi Takaba, Saori Maki-Yonekura, Koji Yonekura

    Journal of structural biology　211　(2)　107549-107549　2020/08/01

    DOI： 10.1016/j.jsb.2020.107549 　

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    A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12-20 rotational series from ~-68° to ~68° at a rotation speed of 1°/s in one hour without human supervision.

  65. Tidy up cryo-EM sample grids with 3D printed tools. International-journal Peer-reviewed

    Tasuku Hamaguchi, Koji Yonekura

    Journal of structural biology　209　(1)　107414-107414　2020/01/01

    DOI： 10.1016/j.jsb.2019.107414 　

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    Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases. These tools are made of a plastic fiber using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas. A grid case stacker organizes many frozen-hydrated cryo-EM grids and the stackers can be piled up inside a standard 50 mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. Blueprints of the tools named CryoGridTools are available from a GitHub site.

  66. Understanding hydrogen-bonding structures of molecular crystals via electron and NMR nanocrystallography. International-journal Peer-reviewed

    Candelaria Guzmán-Afonso, You-Lee Hong, Henri Colaux, Hirofumi Iijima, Akihiro Saitow, Takuma Fukumura, Yoshitaka Aoyama, Souhei Motoki, Tetsuo Oikawa, Toshio Yamazaki, Koji Yonekura, Yusuke Nishiyama

    Nature communications　10　(1)　3537-3537　2019/08/06

    DOI： 10.1038/s41467-019-11469-2 　

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    Understanding hydrogen-bonding networks in nanocrystals and microcrystals that are too small for X-ray diffractometry is a challenge. Although electron diffraction (ED) or electron 3D crystallography are applicable to determining the structures of such nanocrystals owing to their strong scattering power, these techniques still lead to ambiguities in the hydrogen atom positions and misassignments of atoms with similar atomic numbers such as carbon, nitrogen, and oxygen. Here, we propose a technique combining ED, solid-state NMR (SSNMR), and first-principles quantum calculations to overcome these limitations. The rotational ED method is first used to determine the positions of the non-hydrogen atoms, and SSNMR is then applied to ascertain the hydrogen atom positions and assign the carbon, nitrogen, and oxygen atoms via the NMR signals for 1H, 13C, 14N, and 15N with the aid of quantum computations. This approach elucidates the hydrogen-bonding networks in L-histidine and cimetidine form B whose structure was previously unknown.

  67. 微小な結晶から構造を見る ― 電子回析を用いた低分子医薬品の結晶構造解析 ― Invited

    西山裕介, 米倉功治

    現代化学　32-37　2019/08

  68. Imaging Theory after the cryo-EM Revolutionary Advancement Invited Peer-reviewed

    Koji Yonekura

    54　(2)　91-97　2019/08

    DOI： 10.11410/kenbikyo.54.2_91 　

  69. CryoTEM with a Cold Field Emission Gun That Moves Structural Biology into a New Stage Invited

    Takayuki Kato, Fumiaki Makino, Takanori Nakane, Naoya Terahara, Takeshi Kaneko, Yuko Shimizu, Sohei Motoki, Isamu Ishikawa, Koji Yonekura, Keiichi Namba

    Microscopy and Microanalysis　25　(S2)　998-999　2019/08

    Publisher: Cambridge University Press (CUP)

    DOI： 10.1017/s1431927619005725 　

    ISSN： 1431-9276

    eISSN： 1435-8115

  70. Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus. International-journal Peer-reviewed

    Marie Kubota, Rei Matsuoka, Tateki Suzuki, Koji Yonekura, Yusuke Yanagi, Takao Hashiguchi

    Journal of virology　93　(15)　2019/08/01

    DOI： 10.1128/JVI.00344-19 　

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    Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid as a receptor. However, given the MuV tropism toward glandular tissues and the central nervous system, an additional glycan motif(s) may also serve as a receptor. Here, we performed a large-scale glycan array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN proteins bound to newly identified glycan motifs, sialyl LewisX (SLeX) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLeX and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLeX and GM2-glycan have several unique interactions compared with those of 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLeX at the second and third carbohydrates from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLeX, or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The α2,3-sialylated oligosaccharides, such as SLeX and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism.IMPORTANCE Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl LewisX and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV.

  71. A new cryo-EM system for single particle analysis. International-journal Peer-reviewed

    Tasuku Hamaguchi, Saori Maki-Yonekura, Hisashi Naitow, Yoshinori Matsuura, Tetsuya Ishikawa, Koji Yonekura

    Journal of structural biology　207　(1)　40-48　2019/07/01

    DOI： 10.1016/j.jsb.2019.04.011 　

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    A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.

  72. A new cryo-EM system for electron 3D crystallography by eEFD. International-journal Peer-reviewed

    Koji Yonekura, Tetsuya Ishikawa, Saori Maki-Yonekura

    Journal of structural biology　206　(2)　243-253　2019/05/01

    DOI： 10.1016/j.jsb.2019.03.009 　

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    A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.

  73. Correction: Hexameric and pentameric complexes of the ExbBD energizer in the Ton system. International-journal Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Rei Matsuoka, Yoshiki Yamashita, Hirofumi Shimizu, Maiko Tanaka, Fumie Iwabuki

    eLife　7　2018/05/04

    DOI： 10.7554/eLife.37833 　

  74. Ionic scattering factors of atoms that compose biological molecules. International-journal Peer-reviewed

    Koji Yonekura, Rei Matsuoka, Yoshiki Yamashita, Tsutomu Yamane, Mitsunori Ikeguchi, Akinori Kidera, Saori Maki-Yonekura

    IUCrJ　5　(Pt 3)　348-353　2018/05/01

    DOI： 10.1107/S2052252518005237 　

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    Ionic scattering factors of atoms that compose biological molecules have been computed by the multi-configuration Dirac-Fock method. These ions are chemically unstable and their scattering factors had not been reported except for O-. Yet these factors are required for the estimation of partial charges in protein molecules and nucleic acids. The electron scattering factors of these ions are particularly important as the electron scattering curves vary considerably between neutral and charged atoms in the spatial-resolution range explored in structural biology. The calculated X-ray and electron scattering factors have then been parameterized for the major scattering curve models used in X-ray and electron protein crystallography and single-particle cryo-EM. The X-ray and electron scattering factors and the fitting parameters are presented for future reference.

  75. Hexameric and pentameric complexes of the ExbBD energizer in the Ton system. International-journal Peer-reviewed

    Saori Maki-Yonekura, Rei Matsuoka, Yoshiki Yamashita, Hirofumi Shimizu, Maiko Tanaka, Fumie Iwabuki, Koji Yonekura

    eLife　7　2018/04/17

    DOI： 10.7554/eLife.35419 　

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    Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

  76. Refinement of cryo-EM structures using scattering factors of charged atoms Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura

    Journal of Applied Crystallography　49　(5)　1517-1523　2016/10/01

    Publisher: International Union of Crystallography (IUCr)

    DOI： 10.1107/s1600576716011274 　

    eISSN： 1600-5767

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    This paper reports a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-electron microscopy (cryo-EM) maps. The ScatCurve package developed here supports various curve models for parameterization of scattering factors and the parameter tables can be implemented in major refinement programs in structural biology. Partial charge values of charged amino acids in crystal structures were changed in small steps for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, the authors found the electrostatic setting that produces atomic models with improved statistics and better reflects experimental data. Structure refinement for single-particle analysis also benefits from the more accurate analysis and the programs could find wide use for model refinement against cryo-EM maps.

  77. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains. International-journal Peer-reviewed

    Eriko Nango, Shuji Akiyama, Saori Maki-Yonekura, Yuji Ashikawa, Yuko Kusakabe, Elena Krayukhina, Takahiro Maruno, Susumu Uchiyama, Nipawan Nuemket, Koji Yonekura, Madoka Shimizu, Nanako Atsumi, Norihisa Yasui, Takaaki Hikima, Masaki Yamamoto, Yuji Kobayashi, Atsuko Yamashita

    Scientific reports　6　25745-25745　2016/05/10

    DOI： 10.1038/srep25745 　

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    Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states.

  78. Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy. International-journal Peer-reviewed

    Yuki Takayama, Koji Yonekura

    Acta crystallographica. Section A, Foundations and advances　72　(Pt 2)　179-89　2016/03

    DOI： 10.1107/S2053273315023980 　

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    Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

  79. Cryogenic coherent x-ray diffraction imaging for biological non-crystalline particles using the KOTOBUKI-1 diffraction apparatus at SACLA Peer-reviewed

    Tomotaka Oroguchi, Yuki Sekiguchi, Amane Kobayashi, Yu Masaki, Asahi Fukuda, Saki Hashimoto, Masayoshi Nakasako, Yuichi Ichikawa, Hitoshi Kurumizaka, Mitsuhiro Shimizu, Yayoi Inui, Sachihiro Matsunaga, Takayuki Kato, Keiichi Namba, Keiichi Yamaguchi, Kazuo Kuwata, Hiroshi Kameda, Naoya Fukui, Yasushi Kawata, Takashi Kameshima, Yuki Takayama, Koji Yonekura, Masaki Yamamoto

    Journal of Physics B: Atomic, Molecular and Optical Physics　48　(18)　2015/07/29

    Publisher: Institute of Physics Publishing

    DOI： 10.1088/0953-4075/48/18/184003 　

    ISSN： 1361-6455 0953-4075

  80. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges. International-journal Peer-reviewed

    Koji Yonekura, Kazuyuki Kato, Mitsuo Ogasawara, Masahiro Tomita, Chikashi Toyoshima

    Proceedings of the National Academy of Sciences of the United States of America　112　(11)　3368-73　2015/03/17

    DOI： 10.1073/pnas.1500724112 　

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    Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

  81. Signal enhancement and Patterson-search phasing for high-spatial-resolution coherent X-ray diffraction imaging of biological objects. International-journal Peer-reviewed

    Yuki Takayama, Saori Maki-Yonekura, Tomotaka Oroguchi, Masayoshi Nakasako, Koji Yonekura

    Scientific reports　5　8074-8074　2015/01/28

    DOI： 10.1038/srep08074 　

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    In this decade coherent X-ray diffraction imaging has been demonstrated to reveal internal structures of whole biological cells and organelles. However, the spatial resolution is limited to several tens of nanometers due to the poor scattering power of biological samples. The challenge is to recover correct phase information from experimental diffraction patterns that have a low signal-to-noise ratio and unmeasurable lowest-resolution data. Here, we propose a method to extend spatial resolution by enhancing diffraction signals and by robust phasing. The weak diffraction signals from biological objects are enhanced by interference with strong waves from dispersed colloidal gold particles. The positions of the gold particles determined by Patterson analysis serve as the initial phase, and this dramatically improves reliability and convergence of image reconstruction by iterative phase retrieval. A set of calculations based on current experiments demonstrates that resolution is improved by a factor of two or more.

  82. Single-shot three-dimensional structure determination of nanocrystals with femtosecond X-ray free-electron laser pulses. International-journal Peer-reviewed

    Rui Xu, Huaidong Jiang, Changyong Song, Jose A Rodriguez, Zhifeng Huang, Chien-Chun Chen, Daewoong Nam, Jaehyun Park, Marcus Gallagher-Jones, Sangsoo Kim, Sunam Kim, Akihiro Suzuki, Yuki Takayama, Tomotaka Oroguchi, Yukio Takahashi, Jiadong Fan, Yunfei Zou, Takaki Hatsui, Yuichi Inubushi, Takashi Kameshima, Koji Yonekura, Kensuke Tono, Tadashi Togashi, Takahiro Sato, Masaki Yamamoto, Masayoshi Nakasako, Makina Yabashi, Tetsuya Ishikawa, Jianwei Miao

    Nature communications　5　4061-4061　2014/06/05

    DOI： 10.1038/ncomms5061 　

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    Conventional three-dimensional (3D) structure determination methods require either multiple measurements at different sample orientations or a collection of serial sections through a sample. Here we report the experimental demonstration of single-shot 3D structure determination of an object; in this case, individual gold nanocrystals at ~5.5 nm resolution using ~10 fs X-ray free-electron laser pulses. Coherent diffraction patterns are collected from high-index-faceted nanocrystals, each struck by an X-ray free-electron laser pulse. Taking advantage of the symmetry of the nanocrystal and the curvature of the Ewald sphere, we reconstruct the 3D structure of each nanocrystal from a single-shot diffraction pattern. By averaging a sufficient number of identical nanocrystals, this method may be used to determine the 3D structure of nanocrystals at atomic resolution. As symmetry exists in many virus particles, this method may also be applied to 3D structure studies of such particles at nanometer resolution on femtosecond time scales.

  83. 2P309 Cryogenic coherent X-ray diffraction imaging of cellular organelle particles(27. Bioimaging,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

    Sekiguchi Yuki, Kobayashi Amane, Hashimoto Saki, Oroguchi Tomotaka, Nakasako Masayoshi, Takayama Yuki, Yonekura Koji, Yamamoto Masaki, Inui Yayoi, Matsunaga Sachihiro, Ichikawa Yuichi, Kurumizaka Hitoshi, Shimizu Mitsuhiro

    Seibutsu Butsuri　54　(1)　S246　2014

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.54.S246_3 　

  84. Coherent X-ray Diffraction Imaging of Non-Crystalline Particles using X-ray Free Electron Laser

    NAKASAKO Masayoshi, OROGUCHI Tomotaka, SEKIGUCHI Yuki, KOBAYASHI Amane, HASHIMOTO Saki, SHIRAHAMA Keiya, YAMAMOTO Masaki, TAKAYAMA Yuki, YONEKURA Koji, MAKI-YONEKURA Saori, HIKIMA Takaaki, TAKAHASHI Yukio, SUZUKI Akihiro, MATSUNAGA Sachihiro, INUI Yayoi, TONO Kensuke, KAMESHIMA Takashi, JOTI Yasumasa, INUBUSHI Yuichi, HOSHI Takahiko

    X-RAYS　56　(1)　27-35　2014

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.56.27 　

    ISSN： 0369-4585

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    Coherent X-ray diffraction imaging（CXDI）is a lens-less imaging technique that can visualize the structures of non-crystalline particles with micro- to sub-micrometer dimensions. In CXDI experiments, spatially isolated particles are irradiated by X-ray beam with high transverse coherence. Projection images of the particles along the incident X-ray direction are then directly reconstructed from the diffraction amplitude using a phase retrieval algorithm. Here we report CXDI experiments carried out at the X-ray free electron laser（XFEL）facility SACLA, and describe the theoretical background of CXDI, development of a diffractometer and data processing.

  85. 3P310 Signal enhancement and Patterson-search phasing for highspatial-resolution coherent X-ray diffraction imaging of biological objects(27. Bioimaging,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

    Takayama Yuki, Maki-Yonekura Saori, Oroguchi Tomotaka, Nakasako Masayoshi, Yonekura Koji

    Seibutsu Butsuri　54　(1)　S300　2014

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.54.S300_4 　

  86. Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus. International-journal Peer-reviewed

    Raymond H J Staals, Yoshihiro Agari, Saori Maki-Yonekura, Yifan Zhu, David W Taylor, Esther van Duijn, Arjan Barendregt, Marnix Vlot, Jasper J Koehorst, Keiko Sakamoto, Akiko Masuda, Naoshi Dohmae, Peter J Schaap, Jennifer A Doudna, Albert J R Heck, Koji Yonekura, John van der Oost, Akeo Shinkai

    Molecular cell　52　(1)　135-145　2013/10/10

    DOI： 10.1016/j.molcel.2013.09.013 　

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    The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

  87. KOTOBUKI-1 apparatus for cryogenic coherent X-ray diffraction imaging. International-journal Peer-reviewed

    Masayoshi Nakasako, Yuki Takayama, Tomotaka Oroguchi, Yuki Sekiguchi, Amane Kobayashi, Keiya Shirahama, Masaki Yamamoto, Takaaki Hikima, Koji Yonekura, Saori Maki-Yonekura, Yoshiki Kohmura, Yuichi Inubushi, Yukio Takahashi, Akihiro Suzuki, Sachihiro Matsunaga, Yayoi Inui, Kensuke Tono, Takashi Kameshima, Yasumasa Joti, Takahiko Hoshi

    The Review of scientific instruments　84　(9)　093705-093705　2013/09

    DOI： 10.1063/1.4822123 　

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    We have developed an experimental apparatus named KOTOBUKI-1 for use in coherent X-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. For cryogenic specimen stage with small positional fluctuation for a long exposure time of more than several minutes, we here use a cryogenic pot cooled by the evaporation cooling effect for liquid nitrogen. In addition, a loading device is developed to bring specimens stored in liquid nitrogen to the specimen stage in vacuum. The apparatus allows diffraction data collection for frozen-hydrated specimens at 66 K with a positional fluctuation of less than 0.4 μm and provides an experimental environment to easily exchange specimens from liquid nitrogen storage to the specimen stage. The apparatus was developed and utilized in diffraction data collection of non-crystalline particles with dimensions of μm from material and biological sciences, such as metal colloid particles and chloroplast, at BL29XU of SPring-8. Recently, it has been applied for single-shot diffraction data collection of non-crystalline particles with dimensions of sub-μm using X-ray free electron laser at BL3 of SACLA.

  88. 1P291 Humidity-controlled preparation of frozen-hydrated biological samples for cryogenic coherent X-ray diffraction imaging using XFEL(27. Bioimaging,Poster)

    Takayama Yuki, Nakasako Masayoshi, Oroguchi Tomotaka, Sekiguchi Yuki, Kobayashi Amane, Yamamoto Masaki, Yonekura Koji, Hikima Takaaki, Maki-Yonekura Saori, Takahashi Yukio, Suzuki Akihiro, Matsunaga Sachihiro, Inui-Tsujimoto Yayoi, Kato Shoichi, Hoshi Takahiko

    Seibutsu Butsuri　53　(1)　S154　2013

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.53.S154_2 　

  89. Post-transcriptional regulator Hfq binds catalase HPII: crystal structure of the complex. International-journal Peer-reviewed

    Koji Yonekura, Masahiro Watanabe, Yuko Kageyama, Kunio Hirata, Masaki Yamamoto, Saori Maki-Yonekura

    PloS one　8　(11)　e78216　2013

    DOI： 10.1371/journal.pone.0078216 　

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    We report a crystal structure of Hfq and catalase HPII from Escherichia coli. The post-transcriptional regulator Hfq plays a key role in the survival of bacteria under stress. A small non-coding RNA (sRNA) DsrA is required for translation of the stationary phase sigma factor RpoS, which is the central regulator of the general stress response. Hfq facilitates efficient translation of rpoS mRNA, which encodes RpoS. Hfq helps in the function of other specific proteins involved in RNA processing, indicating its versatility in the cell. However, structural information regarding its interactions with partners is missing. Here we obtained crystals of Hfq and HPII complexes from cell lysates following attempts to overexpress a foreign membrane protein. HPII is one of two catalases in E. coli and its mRNA is transcribed by an RNA polymerase holoenzyme containing RpoS, which in turn is under positive control of small non-coding RNAs and of the RNA chaperone Hfq. This sigma factor is known to have a pronounced effect on the expression of HPII. The crystal structure reveals that a Hfq hexamer binds each subunit of a HPII tetramer. Each subunit of the Hfq hexamer exhibits a unique binding mode with HPII. The hexamer of Hfq interacts via its distal surface. The proximal and distal surfaces are known to specifically bind different sRNAs, and binding of HPII could affect Hfq function. Hfq-HPII complexation has no effect on catalase HPII activity.

  90. 2D1522 Cryogenic Coherent X-ray Diffraction Imaging of non-crystalline particles at SACLA(Measurements,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)

    Nakasako Masayoshi, Takayama Yuki, Oroguchi Tomotaka, Sekiguchi Yuki, Yamamoto Masaki, Yonekura Koji, Hikima Takaaki, Maki-Yonekura Saori, Takahashi Yukio, Suzuki Akihiro, Matsunaga Sachihiro, Kato Shoichi, Hoshi Takahiko

    Seibutsu Butsuri　52　S44-S45　2012

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.52.S44_6 　

  91. Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation. International-journal Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Michio Homma

    Journal of bacteriology　193　(15)　3863-70　2011/08

    DOI： 10.1128/JB.05021-11 　

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    The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.

  92. Characterization of the yeast telomere nucleoprotein core: Rap1 binds independently to each recognition site. International-journal Peer-reviewed

    Tanya L Williams, Daniel L Levy, Saori Maki-Yonekura, Koji Yonekura, Elizabeth H Blackburn

    The Journal of biological chemistry　285　(46)　35814-24　2010/11/12

    DOI： 10.1074/jbc.M110.170167 　

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    At the core of Saccharomyces cerevisiae telomeres is an array of tandem telomeric DNA repeats bound site-specifically by multiple Rap1 molecules. There, Rap1 orchestrates the binding of additional telomere-associated proteins and negatively regulates both telomere fusion and length homeostasis. Using electron microscopy, viscosity, and light scattering measurements, we show that purified Rap1 is a monomer in solution that adopts a ringlike or C shape with a central cavity. Rap1 could orchestrate telomere function by binding multiple telomere array sites through either cooperative or independent mechanisms. To determine the mechanism, we analyze the distribution of Rap1 monomers on defined telomeric DNA arrays. This analysis clearly indicates that Rap1 binds independently to each nonoverlapping site in an array, regardless of the spacing between sites, the total number of sites, the affinity of the sites for Rap1, and over a large concentration range. Previous experiments have not clearly separated the effects of affinity from repeat spacing on telomere function. We clarify these results by testing in vivo the function of defined telomere arrays containing the same Rap1 binding site separated by spacings that were previously defined as low or high activity. We find that Rap1 binding affinity in vitro correlates with the ability of telomeric repeat arrays to regulate telomere length in vivo. We suggest that Rap1 binding to multiple sites in a telomere array does not, by itself, promote formation of a more energetically stabile complex.

  93. Conformational change of flagellin for polymorphic supercoiling of the flagellar filament. International-journal Peer-reviewed

    Saori Maki-Yonekura, Koji Yonekura, Keiichi Namba

    Nature structural & molecular biology　17　(4)　417-22　2010/04

    DOI： 10.1038/nsmb.1774 　

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    The bacterial flagellar filament is a helical propeller rotated by the flagellar motor for bacterial locomotion. The filament is a supercoiled assembly of a single protein, flagellin, and is formed by 11 protofilaments. For bacterial taxis, the reversal of motor rotation switches the supercoil between left- and right-handed, both of which arise from combinations of two distinct conformations and packing interactions of the L-type and R-type protofilaments. Here we report an atomic model of the L-type straight filament by electron cryomicroscopy and helical image analysis. Comparison with the R-type structure shows interesting features: an orientation change of the outer core domains (D1) against the inner core domains (D0) showing almost invariant orientation and packing, a conformational switching within domain D1, and the conformational flexibility of domains D0 and D1 with their spoke-like connection for tight molecular packing.

  94. Cryo-Electron Microscopy of Biological Macromolecular Structures

    YONEKURA Koji

    X-RAYS　52　(1)　56-61　2010/02/28

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.52.56 　

    ISSN： 0369-4585

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    There are many huge macromolecular complexes in living organisms. They are often hard to crystallize because of their size, complexity and heterogeneity. Cryo-electron microscopy (cryo-EM) is a suitable method to analyze the structures of such biological macromolecules, because it can be applied to various forms of samples, e.g. two-dimensional crystal, helical assembly, spherical virus, dispersed particle, cell organelle and cell, although attainable resolution depends on the system. In this review, I introduce these techniques and examples of the structure analysis, and briefly review the perspective of cryo-EM.

  95. Pleomorphic configuration of the trimeric capsid proteins of Rice dwarf virus that allows formation of both the outer capsid and tubular crystals. International-journal Peer-reviewed

    Kenji Iwasaki, Naoyuki Miyazaki, Lena Hammar, Yafeng Zhu, Toshihiro Omura, Bomu Wu, Fredrik Sjöborg, Koji Yonekura, Kazuyoshi Murata, Keiichi Namba, Donald L Caspar, Yoshinori Fujiyoshi, R Holland Cheng

    Journal of molecular biology　383　(1)　252-65　2008/10/31

    DOI： 10.1016/j.jmb.2008.08.021 　

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    In the double-shelled capsid of Phytoreovirus, the outer capsid attaches firmly to the 3-fold axes of the T=1 core. It then forms a T=13 lattice via lateral interactions among the P8 trimers (Wu et al., 2000, Virology 271, 18-25). Purified P8 molecules also assemble into hexagonal monolayers as well as tubular crystals. To explore the mechanisms of formation of these structures, the configurations of P8 trimers were compared and verified in particles of Rice dwarf virus and in tubular crystals (tubes) whose structure was determined by cryoelectron microscopy using helical reconstruction technique. Remarkable variations in intertrimer contacts were observed in the tubes and in the surface lattice of Rice dwarf virus capsid. Superposition of the atomic structure of P8 trimers in the structures analyzed by cryoelectron microscopy allowed us to identify groups of specific and stable interactions, some of which were preserved in the tubes and the quasi-equivalent T=13 icosahedral lattice of the virion's shell. The flexible nature of the binding between P8 trimers, created via electrostatic interactions that hold radially inward, appears to allow the outer-capsid P8 trimers to envelop the ragged surface of the core, forming the double shell of an intact viral particle.

  96. Electron digital imaging toward high-resolution structure analysis of biological macromolecules. International-journal Peer-reviewed

    Saori Maki-Yonekura, Koji Yonekura

    Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada　14　(4)　362-9　2008/08

    DOI： 10.1017/S1431927608080665 　

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    Digital imaging has been applied to structure analysis of biological macromolecules in combination with electron energy filtering. Energy filtering can improve the image contrast of frozen-hydrated specimens, but needs a high-sensitivity imaging device instead of photographic film, because of a decrease in electrons after filtration. Here, a lens-coupled slow-scan charge-coupled device (SSCCD) camera with a post-column-type energy filter were examined to image bacterial flagellar filaments embedded in ice. We first measured the modulation transfer function of this camera and showed the remarkable improvement, compared to other fiber-coupled SSCCD cameras. The 3D structure calculated at approximately 7-angstroms resolution clearly resolves alpha-helices. Furthermore, filtered datasets recorded on the SSCCD camera with liquid-nitrogen and liquid-helium cooling were compared with the previous unfiltered one on film with liquid-helium cooling. This report describes the suitability of digital imaging with energy filtering for higher-resolution structure studies from its practical application.

  97. 2P-105 Structure analysis of S. cerevisiae Rap1-telomeric DNA complex by Electron microscopy(The 46th Annual Meeting of the Biophysical Society of Japan)

    Maki-Yonekura Saori, Williams Tanya L., Blackburn Elizabeth H., Yonekura Koji

    Seibutsu Butsuri　48　S91　2008

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.48.S91_4 　

  98. 2S3-5 Cryo-electron microscopy of the bacterial flagellar motor(2S3 Structure and functional mechanism of the bacterial flagellar motor,The 46th Annual Meeting of the Biophysical Society of Japan)

    Yonekura Koji

    Seibutsu Butsuri　48　S9　2008

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.48.S9_4 　

  99. Structure determination of tubular crystals of membrane proteins. IV. Distortion correction and its combined application with real-space averaging and solvent flattening. International-journal Peer-reviewed

    Koji Yonekura, Chikashi Toyoshima

    Ultramicroscopy　107　(12)　1141-58　2007/11

    ISSN： 0304-3991

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    A method for correction of three-dimensional distortions has been developed for helical assemblies and applied to tubular crystals of Ca2+-ATPase. This method approximates distorted helical particles with short straight segments of different orientation parameters, which are determined by fitting them to the reference data in reciprocal space. Thus, the method follows Beroukhim and Unwin [Ultramicroscopy 70 (1997) 57], but is more extended to achieve better alignment and to cope with images of poor S/N ratio. Substantial improvements were achieved by dividing the reference image into the segments of optimal lengths in exactly the same way as the test, and treating the distortions in the near and far sides of a helical particle separately. The improvement was further enhanced when combined with real-space averaging [Yonekura, Toyoshima, Ultramicroscopy 84 (2000) 15] and solvent flattening [Yonekura, Toyoshima, Ultramicroscopy 84 (2000) 29], and most pronounced when all these three were applied iteratively.

  100. Electron energy filtering significantly improves amplitude contrast of frozen-hydrated protein at 300kV. International-journal Peer-reviewed

    Koji Yonekura, Michael B Braunfeld, Saori Maki-Yonekura, David A Agard

    Journal of structural biology　156　(3)　524-36　2006/12

    ISSN： 1047-8477

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    The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9+/-1.9% and 2.7+/-1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5+/-2.0% and 5.8+/-1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least approximately 25A resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to approximately 15A. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8A by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.

  101. Electron Cryomicroscopy of Bacterial Flagellar Structures Invited

    K Yonekura

    Microscopy and Microanalysis　12　(S02)　28-29　2006/08

    Publisher: Cambridge University Press (CUP)

    DOI： 10.1017/s1431927606068188 　

    ISSN： 1431-9276

    eISSN： 1435-8115

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    Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

  102. Switch interactions control energy frustration and multiple flagellar filament structures. International-journal Peer-reviewed

    Akio Kitao, Koji Yonekura, Saori Maki-Yonekura, Fadel A Samatey, Katsumi Imada, Keiichi Namba, Nobuhiro Go

    Proceedings of the National Academy of Sciences of the United States of America　103　(13)　4894-9　2006/03/28

    ISSN： 0027-8424

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    Bacterial flagellar filament is a macromolecular assembly consisting of a single protein, flagellin. Bacterial swimming is controlled by the conformational transitions of this filament between left- and right-handed supercoils induced by the flagellar motor torque. We present a massive molecular dynamics simulation that was successful in constructing the atomic-level supercoil structures consistent with various experimental data and further in elucidating the detailed underlying molecular mechanisms of the polymorphic supercoiling. We have found that the following three types of interactions are keys to understanding the supercoiling mechanism. "Permanent" interactions are always maintained between subunits in the various supercoil structures. "Sliding" interactions are formed between variable hydrophilic or hydrophobic residue pairs, allowing intersubunit shear without large change in energy. The formation and breakage of "switch" interactions stabilize inter- and intrasubunit interactions, respectively. We conclude that polymorphic supercoiling is due to the energy frustration between them. The transition between supercoils is achieved by a "transform and relax" mechanism: the filament structure is geometrically transformed rapidly and then slowly relaxes to energetically metastable states by rearranging interactions.

  103. Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes. International-journal Peer-reviewed

    Koji Yonekura, Toshiharu Yakushi, Tatsuo Atsumi, Saori Maki-Yonekura, Michio Homma, Keiichi Namba

    Journal of molecular biology　357　(1)　73-81　2006/03/17

    ISSN： 0022-2836

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    A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.

  104. [Structure, function and self-assembly of the bacterial flagellum].

    Katsumi Imada, Fadel A Samatey, Hideyuki Matsunami, Shigehiro Nagashima, Akio Kitao, Koji Yonekura, Saori Maki-Yonekuraz, Keiichi Namba

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme　50　(10 Suppl)　1328-34　2005/08

    ISSN： 0039-9450

  105. Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins. International-journal Peer-reviewed

    Motomasa Tanaka, Peter Chien, Koji Yonekura, Jonathan S Weissman

    Cell　121　(1)　49-62　2005/04/08

    ISSN： 0092-8674

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    Efficiency of interspecies prion transmission decreases as the primary structures of the infectious proteins diverge. Yet, a single prion protein can misfold into multiple infectious conformations, and such differences in "strain conformation" also alter infection specificity. Here, we explored the relationship between prion strains and species barriers by creating distinct synthetic prion forms of the yeast prion protein Sup35. We identified a strain conformation of Sup35 that allows transmission from the S. cerevisiae (Sc) Sup35 to the highly divergent C. albicans (Ca) Sup35 both in vivo and in vitro. Remarkably, cross-species transmission leads to a novel Ca strain that in turn can infect the Sc protein. Structural studies reveal strain-specific conformational differences in regions of the prion domain that are involved in intermolecular contacts. Our findings support a model whereby strain conformation is the critical determinant of cross-species prion transmission while primary structure affects transmission specificity by altering the spectrum of preferred amyloid conformations.

  106. Building the atomic model for the bacterial flagellar filament by electron cryomicroscopy and image analysis. International-journal Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Structure (London, England : 1993)　13　(3)　407-12　2005/03

    ISSN： 0969-2126

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    The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a well-ordered helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments, each in either of the two distinct conformations, L- and R-type, for supercoiling. We have been studying the three-dimensional structures of the flagellar filaments by electron cryomicroscopy and recently obtained a density map of the R-type filament up to 4 angstroms resolution from an image data set containing only about 41,000 molecular images. The density map showed the features of the alpha-helical backbone and some large side chains, which allowed us to build the complete atomic model as one of the first atomic models of macromolecules obtained solely by electron microscopy image analysis (Yonekura et al., 2003a). We briefly review the structure and the structure analysis, and point out essential techniques that have made this analysis possible.

  107. Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis. International-journal Peer-reviewed

    Hirofumi Suzuki, Koji Yonekura, Keiichi Namba

    Journal of molecular biology　337　(1)　105-13　2004/03/12

    ISSN： 0022-2836

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    The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor. We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively. Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages.

  108. [Electron cryomicroscopy of the bacterial flagellar nanomachine]. Invited Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme　49　(4)　534-42　2004/03

    ISSN： 0039-9450

  109. 1P056 Supercoil transition of bacterial flagellar filament by molecular dynamics simulation

    Kitao A., Yonekura K., Maki S., Samatey FadelA, Imada K., Namba K., Go N.

    Seibutsu Butsuri　44　S43　2004

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.44.S43_4 　

  110. Domain movements of HAP2 in the cap-filament complex formation and growth process of the bacterial flagellum. International-journal Peer-reviewed

    Saori Maki-Yonekura, Koji Yonekura, Keiichi Namba

    Proceedings of the National Academy of Sciences of the United States of America　100　(26)　15528-33　2003/12/23

    ISSN： 0027-8424

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    The cap at the growing end of the bacterial flagellum is essential for its growth, remaining stably attached while permitting the insertion of flagellin transported from the cytoplasm through the narrow central channel. We analyzed the structure of the isolated cap in its frozen hydrated state by electron cryomicroscopy. The 3D density map now shows detailed features of domains and their connections, giving reliable volumes and masses, making assignment of the domains to the amino acid sequence possible. A model of the cap-filament complex built with an atomic model of the filament allows a quantitative analysis of the cap domain movements on cap binding and rotation that promotes the efficient self assembly of flagellin during the filament growth process.

  111. GUI programs for processing individual images in early stages of helical image reconstruction—for high-resolution structure analysis Peer-reviewed

    Koji Yonekura, Chikashi Toyoshima, Saori Maki-Yonekura, Keiichi Namba

    Journal of Structural Biology　144　(1-2)　184-194　2003/10

    Publisher: Elsevier BV

    DOI： 10.1016/j.jsb.2003.09.034 　

    ISSN： 1047-8477

  112. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. International-journal Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Nature　424　(6949)　643-50　2003/08/07

    eISSN： 1476-4687

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    The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments in two distinct conformations, L- and R-type, for supercoiling. The X-ray crystal structure of a flagellin fragment lacking about 100 terminal residues revealed the protofilament structure, but the full filament structure is still essential for understanding the mechanism of supercoiling and polymerization. Here we report a complete atomic model of the R-type filament by electron cryomicroscopy. A density map obtained from image data up to 4 A resolution shows the feature of alpha-helical backbone and some large side chains. The atomic model built on the map reveals intricate molecular packing and an alpha-helical coiled coil formed by the terminal chains in the inner core of the filament, with its intersubunit hydrophobic interactions having an important role in stabilizing the filament.

  113. ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC. International-journal Peer-reviewed

    Fumio Hayashi, Hirofumi Suzuki, Ryo Iwase, Tatsuya Uzumaki, Asako Miyake, Jian-Ren Shen, Katsumi Imada, Yukio Furukawa, Koji Yonekura, Keiichi Namba, Masahiro Ishiura

    Genes to cells : devoted to molecular & cellular mechanisms　8　(3)　287-96　2003/03

    ISSN： 1356-9597

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    BACKGROUND: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP-binding Walker's motif As, and mutations in these regions affect the clock oscillations. RESULTS: ATP induced the hexamerization of KaiC. The Km value for the ATP for the hexamerization was 1.9 micro m. Triphosphate nucleotides bound to the two Walker's motif As, and their binding functioned cooperatively for the hexamerization. An unhydrolysable substrate, 5'-adenylylimidodiphosphate (AMPPNP), also induced the hexamerization, indicating that nucleotide binding, but not its hydrolysis, is essential for the hexamerization. Mutations in each of the two Walker's motif As that affect the clock phenotype increased the Km value for ATP and inhibited the hexamerization. Thus, the KaiC hexamerization seems to be necessary for its clock function. The KaiC hexamer has the shape of a hexagonal pot with a diameter and height of approximately 100 A and with a relatively large cavity (73 A deep and 18-34 A wide) inside. This pot-shaped structure suggests that KaiC functions in a similar manner to F1-ATPase, helicase or ATP-dependent protease/chaperon, all of which have dynamic activities inside the central cavity of their hexameric rings. CONCLUSION: ATP-induced KaiC hexamerization is necessary for the clock function of KaiC.

  114. Three dimensional structure of FliI hexameric ring, export engine of the bacterial flagellum

    Suzuki H., Minamino T., Yonekura K., Namba K.

    Seibutsu Butsuri　43　S107　2003

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.43.S107_1 　

  115. Massive molecular dynamics simulation to invesitigate structure change of bacterial flagellar filament

    Kitao A., Yonekura K., Maki S., Samatey FadelA, Imada K., Namba K., Go N.

    Seibutsu Butsuri　43　S11　2003

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.43.S11_1 　

  116. Refinement of purification of PomAB, ion selector proteins of Na^+-driven flagella motor of Vibrio

    Atsumi T., Yakushi T., Yonekura K., Furukawa Y., Nanba K., Homma M.

    Seibutsu Butsuri　43　S132　2003

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.43.S132_4 　

  117. Observation of a Na^+-driven motor component, PomA/B, by electron cryomicroscopy

    Yonekura K., Yakushi T., Atsumi T., Maki-Yonekura S., Matsunami H., Homma M., Namba K.

    Seibutsu Butsuri　43　S132　2003

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.43.S132_3 　

  118. Comparison of the L- and R-type flagellin structure in the filament revealed by electron cryomicroscopy

    Maki S., Yonekura K., Namba K.

    Seibutsu Butsuri　43　S150　2003

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.43.S150_4 　

  119. Quantitative comparison of zero-loss and conventional electron diffraction from two-dimensional and thin three-dimensional protein crystals. International-journal Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Biophysical journal　82　(5)　2784-97　2002/05

    ISSN： 0006-3495

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    The scattering cross-section of atoms in biological macromolecules for both elastically and inelastically scattered electrons is approximately 100,000 times larger than that for x-ray. Therefore, much smaller (<1 microm) and thinner (<0.01 microm) protein crystals than those used for x-ray crystallography can be used to analyze the molecular structures by electron crystallography. But, inelastic scattering is a serious problem. We examined electron diffraction data from thin three-dimensional (3-D) crystals (600-750 A thick) and two-dimensional (2-D) crystals (approximately 60 A thick), both at 93 K, with an energy filtering electron microscope operated at an accelerating voltage of 200 kV. Removal of inelastically scattered electrons significantly improved intensity data statistics and R(Friedel) factor in every resolution range up to 3-A resolution. The effect of energy filtering was more prominent for thicker crystals but was significant even for thin crystals. These filtered data sets showed better intensity statistics even in comparison with data sets collected at 4 K and an accelerating voltage of 300 kV without energy filtering. Thus, the energy filter will be an effective and important tool in the structure analysis of thin 3-D and 2-D crystals, particularly when data are collected at high tilt angle.

  120. Growth mechanism of the bacterial flagellar filament. International-journal Invited Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Research in microbiology　153　(4)　191-7　2002/05

    ISSN： 0923-2508

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    The growth of the bacterial flagellar filament occurs at its distal end by self-assembly of flagellin transported from the cytoplasm through the narrow central channel of the flagellum. The cap at the growing end is essential for its growth, remaining stably attached while permitting the flagellin insertion. The structure of the cap-filament analyzed by electron cryomicroscopy suggested a cap rotation mechanism to promote the flagellin self-assembly.

  121. 2C1615 Active conformation of FliI, type III export engine of bacterial flagellum

    Suzuki H., Minamino T., Yonekura K., Namba K.

    Seibutsu Butsuri　42　(2)　S106　2002

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.42.S106_2 　

  122. Erratum: Growth mechanism of the bacterial flagellar filament (Research in Microbiology (2002) 153 (191-197) PII: S0923250802013086) Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Research in Microbiology　153　(6)　385　2002

    DOI： 10.1016/S0923-2508(02)01349-9 　

    ISSN： 0923-2508

  123. 3H1500 Structure analysis of bacterial flagellar filament by X-ray fiber diffraction

    Hasegawa K., Imada K., Maki-Yonekura S., Yonekura K., Samatey Fadel, Yamashita I., Namba K.

    Seibutsu Butsuri　42　(2)　S171　2002

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.42.S171_3 　

  124. 2C1415 Atomic model of the bacterial flagellar filament revealed by electron cryomicroscopy

    Yonekura K., Maki-Yonekura S., Namba K.

    Seibutsu Butsuri　42　(2)　S104　2002

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.42.S104_3 　

  125. 2C1400 Structure of the L- and R-type straight flagellar filament from Salmonella by electron cryomicroscopy

    Maki-Yonekura S., Yonekura K., Namba K.

    Seibutsu Butsuri　42　(2)　S104　2002

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.42.S104_2 　

  126. Structure Analysis of the Flagellar Cap–Filament Complex by Electron Cryomicroscopy and Single-Particle Image Analysis Peer-reviewed

    Koji Yonekura, Saori Maki-Yonekura, Keiichi Namba

    Journal of Structural Biology　133　(2-3)　246-253　2001/02

    Publisher: Elsevier BV

    DOI： 10.1006/jsbi.2000.4345 　

    ISSN： 1047-8477

  127. Structure analysis of the rotor core of the bacterial flagellar motor by electron cryo microscopy

    Suzuki H., Yonekura K., Namba K.

    Seibutsu Butsuri　41　S125　2001

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.41.S125_2 　

  128. Interactions between the bacterial flagellar filament and its cap protein, HAP2, revealed by electron cryomicroscopy

    Maki-Yonekura S., Yonekura K., Imada K., Namba K.

    Seibutsu Butsuri　41　S125　2001

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.41.S125_1 　

  129. X-ray crystallographic study of the bacterial flagellar HAP2 cap complex

    Imada K., Maki S., Yonekura K., Samatey Fadel, Nagashima S., Furukawa A., Nammba K.

    Seibutsu Butsuri　41　S126　2001

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.41.S126_3 　

  130. Quantitative analysis of zero-loss electron diffraction patterns from protein crystals

    Yonekura K., Maki-Yonekura S., Namba K.

    Seibutsu Butsuri　41　S109　2001

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.41.S109_2 　

  131. The Bacterial Flagellar Cap as the Rotary Promoter of Flagellin Self-Assembly Peer-reviewed

    K. Yonekura, S. Maki, D. G. Morgan, D. J. DeRosier, F. Vonderviszt, K. Imada, K. Namba

    Science　290　(5499)　2148-2152　2000/12/15

    Publisher: American Association for the Advancement of Science (AAAS)

    DOI： 10.1126/science.290.5499.2148 　

    ISSN： 0036-8075

    eISSN： 1095-9203

    More details Close

    The growth of the bacterial flagellar filament occurs at its distal end by self-assembly of flagellin transported from the cytoplasm through the narrow central channel. The cap at the growing end is essential for its growth, remaining stably attached while permitting the flagellin insertion. In order to understand the assembly mechanism, we used electron microscopy to study the structures of the cap-filament complex and isolated cap dimer. Five leg-like anchor domains of the pentameric cap flexibly adjusted their conformations to keep just one flagellin binding site open, indicating a cap rotation mechanism to promote the flagellin self-assembly. This represents one of the most dynamic movements in protein structures.

  132. Structure determination of tubular crystals of membrane proteins. III. Solvent flattening Peer-reviewed

    Koji Yonekura, Chikashi Toyoshima

    Ultramicroscopy　84　(1-2)　29-45　2000/07

    Publisher: Elsevier BV

    DOI： 10.1016/s0304-3991(00)00008-5 　

    ISSN： 0304-3991

  133. Structure determination of tubular crystals of membrane proteins. II. Averaging of tubular crystals of different helical classes Peer-reviewed

    Koji Yonekura, Chikashi Toyoshima

    Ultramicroscopy　84　(1-2)　15-28　2000/07

    Publisher: Elsevier BV

    DOI： 10.1016/s0304-3991(00)00009-7 　

    ISSN： 0304-3991

  134. Molecular mechanism of self-assembly and polymorphic supercoiling of bacterial flagellar filament based on the atomic structure

    Imada K., Samatey F.A., Hasegawa K., Maki S., Yonekura K., Yamashita I., Namba K.

    Seibutsu Butsuri　40　S144　2000

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.40.S144_3 　

    ISSN： 0582-4052

  135. Rotational assembly-promotion mechanism at the site of flagellin assembly revealed in the flagellar cap structure

    Yonekura Koji, Maki Saori, Morgan David, DeRosier David, Vonderviszt Ferenc, Imada Katsumi, Namba Keiichi

    Seibutsu Butsuri　40　S145　2000

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.40.S145_2 　

    ISSN： 0582-4052

  136. Stabilization of the bacterial flagellar filament by the binding of divalent cations

    Hasegawa K., Yamashita I., Samatey F.A., Imada K., Maki S., Yonekura K., Namba K.

    Seibutsu Butsuri　40　S145　2000

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.40.S145_3 　

    ISSN： 0582-4052

  137. Cylindrically averaged structures of the bacterial flagellar FliF ring and FliF-FliG ring

    Suzuki H., Yonekura K., Namba K.

    Seibutsu Butsuri　40　S146　2000

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.40.S146_1 　

    ISSN： 0582-4052

  138. Structure Analysis of the R-type Straight Flagellar Filament from Salmonella by Electron Cryomicroscopy

    Maki S., Yonekura K., Namba K.

    Seibutsu Butsuri　39　S47　1999

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.39.S47_2 　

    ISSN： 0582-4052

  139. The structure of the filament-HAP2 cap complex of the bacterial flagellum

    Yonekura Koji, Maki Saori, Morgan David G., DeRosier David J., Vonderviszt Ferenc, Imada Katsumi, Namba Keiichi

    Seibutsu Butsuri　39　S46　1999

    Publisher: The Biophysical Society of Japan General Incorporated Association

    DOI： 10.2142/biophys.39.S46_4 　

    ISSN： 0582-4052

  140. A structural feature in the central channel of the bacterial flagellar FliF ring complex is implicated in type III protein export Peer-reviewed

    Hirofumi Suzuki, Koji Yonekura, Kazuyoshi Murata, Teruhisa Hirai, Kenji Oosawa, Keiichi Namba

    Journal of Structural Biology　124　(2-3)　104-114　1998/12/15

    Publisher: Academic Press Inc.

    DOI： 10.1006/jsbi.1998.4048 　

    ISSN： 1047-8477

  141. Cryoelectron microscopy of the calcium pump from sarcoplasmic reticulum: two crystal forms reveal two different conformations Peer-reviewed

    David L. Stokes, Peijun Zhang, Chikashi Toyoshima, Koji Yonekura, Haruo Ogawa, Michael R. Lewis, Dan Shi

    Acta Physiol Scand Suppl.　163　(643)　35-43　1998/08

  142. Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution Peer-reviewed

    Peijun Zhang, Chikashi Toyoshima, Koji Yonekura, N. Michael Green, David L. Stokes

    Nature　392　(6678)　835-839　1998/04

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1038/33959 　

    ISSN： 0028-0836

    eISSN： 1476-4687

  143. The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis Peer-reviewed

    K. Yonekura, D.L. Stokes, H. Sasabe, C. Toyoshima

    Biophysical Journal　72　(3)　997-1005　1997/03

    Publisher: Elsevier BV

    DOI： 10.1016/s0006-3495(97)78752-6 　

    ISSN： 0006-3495

  144. Three-Dimensional Structure of Membrane Proteins.

    Toyoshima Chikashi, Yonekura Koji

    Butsuri　51　(5)　342-346　1996

    Publisher: The Physical Society of Japan

    DOI： 10.11316/butsuri1946.51.342 　

    ISSN： 0029-0181

  145. The Structure of the R-type Straight Flagellar Filament ofSalmonellaat 9 Å Resolution by Electron Cryomicroscopy Peer-reviewed

    Yuko Mimori, Ichiro Yamashita, Kazuyoshi Murata, Yoshinori Fujiyoshi, Koji Yonekura, Chikashi Toyoshima, Keiichi Namba

    Journal of Molecular Biology　249　(1)　69-87　1995/05

    Publisher: Elsevier BV

    DOI： 10.1006/jmbi.1995.0281 　

    ISSN： 0022-2836

  146. Contrast transfer for frozen-hydrated specimens II. Amplitude contrast at very low frequencies Peer-reviewed

    Chikashi Toyoshima, Koji Yonekura, Hiroyuki Sasabe

    Ultramicroscopy　48　(1-2)　165-176　1993/01

    Publisher: Elsevier BV

    DOI： 10.1016/0304-3991(93)90179-2 　

    ISSN： 0304-3991

Show all ︎Show first 5

Misc. 29

  1. Theoretical analysis of optical properties and light harvesting mechanisms in far-red adapted A. marina photosystem I.

    木村明洋, 鬼頭宏任, 青田俊道, 浜口祐, 米倉功治, 川上恵典, 新沢(伊藤)恭子, 井上(菓子野)名津子, 伊福健太郎, 菓子野康浩, 山下栄樹, 伊藤繁

    日本物理学会講演概要集(CD-ROM)　77　(1)　2022

    ISSN： 2189-079X

  2. Competition of Orientational Ordering and Their Controlling in Layered Crystalline Organic Semiconductors, PE-BTBT-Cn's.

    Inoue Satoru, Nikaido Kiyoshi, Higashino Toshiki, Arai Shunto, Tanaka Mutsuo, Horiuchi Sachio, Takaba Kiyofumi, Maki-Yonekura Saori, Yonekura Koji, Segawa Yasutomo, Kumai Reiji, Hasegawa Tatsuo

    JSAP Annual Meetings Extended Abstracts　2021.1　2062-2062　2021/02/26

    Publisher: The Japan Society of Applied Physics

    DOI： 10.11470/jsapmeeting.2021.1.0_2062 　

    eISSN： 2436-7613

  3. Layered Crystallinity and TFT Properties of Unsymmetrically Substituted BTBT Derivatives Having Phenylethynyl Groups Ⅱ

    Inoue Satoru, Higashino Toshiki, Arai Shunto, Tanaka Mutsuo, Horiuchi Sachio, Takaba Toshifumi, Maki-Yonekura Saori, Yonekura Koji, Segawa Yasutomo, Kumai Reiji, Hasegawa Tatsuo

    JSAP Annual Meetings Extended Abstracts　2020.1　2343-2343　2020/02/28

    Publisher: The Japan Society of Applied Physics

    DOI： 10.11470/jsapmeeting.2020.1.0_2343 　

    eISSN： 2436-7613

  4. 【クライオ電子顕微鏡で見えた生命のかたちとしくみ】三次元結晶、単粒子複合解析による膜タンパク質の構造 荷電・プロトン化状態、構造変化・多型を可視化する

    米倉 功治, 眞木 さおり

    実験医学　36　(8)　1333-1338　2018/05

    Publisher: (株)羊土社

    ISSN： 0288-5514

  5. 機能の直接可視化によって解き明かすバクテリア膜蛋白質デバイスの動作原理 プロトン駆動力利用機構のクライオEMと結晶構造解析

    米倉 功治, 眞木 さおり

    日本細菌学雑誌　73　(1)　45-45　2018/02

    Publisher: 日本細菌学会

    ISSN： 0021-4930

    eISSN： 1882-4110

  6. Electron 3D Crystallography of Protein Structures and Refinement of Charges

    59　(2)　88-95　2017/06

    Publisher: 日本結晶学会

    ISSN： 0369-4585

  7. Taste substance binding to the ligand-binding domains of T1r taste receptor heterodimer

    Atsuko Yamashita, Eriko Nango, Shuji Akiyama, Saori Maki-Yonekura, Yuji Ashikawa, Yuko Kusakabe, Elena Krayukhina, Takahiro Maruno, Susumu Uchiyama, Nipawan Nuemket, Koji Yonekura, Madoka Shimizu, Nanako Atsumi, Norihisa Yasui, Takaaki Hikima, Masaki Yamamoto, Yuji Kobayashi

    CHEMICAL SENSES　41　(9)　E176-E177　2016/11

    ISSN： 0379-864X

    eISSN： 1464-3553

  8. 最近の研究と技術 蛋白質の薄い三次元結晶の電子線結晶構造解析と荷電情報の取得

    米倉 功治, 加藤 一幸, 小笠原 光雄, 富田 正弘, 豊島 近

    顕微鏡　50　(3)　201-204　2015/12

    Publisher: (公社)日本顕微鏡学会

    ISSN： 1349-0958

  9. 結晶化に悩む皆様へ 膜蛋白質の電子線三次元結晶構造解析と単粒子解析

    米倉 功治

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集　88回・38回　[2S3-2]　2015/12

    Publisher: (公社)日本生化学会

  10. 低温電子顕微鏡法による蛋白質構造の可視化

    米倉 功治, 眞木 さおり

    感染・炎症・免疫　43　(4)　307-312　2014/01

    Publisher: 鳥居薬品(株)

    ISSN： 0387-1010

  11. Coherent X-Ray Diffraction Imaging of Non-Crystalline Particles

    NAKASAKO Masayoshi, TAKAYAMA Yuki, OROGUCHI Tomotaka, SHIRAHAMA Keiya, SEKIGUCHI Yuki, YAMAMOTO Masaki, YONEKURA Koji, HIKIMA Takaaki, MAKI YONEKURA Saori, TAKAHASHI Yukio, SUZUKI Akihiro, MATSUNAGA Sachihiro, KATO Shoichi, HOSHI Takahiko

    40　(9)　680-686　2012/09/15

    ISSN： 0387-0200

  12. 生体超分子の高分解能構造解析のための電子線イメージングの条件

    米倉 功治

    顕微鏡　45　(4)　243-249　2010/12

    Publisher: (公社)日本顕微鏡学会

    ISSN： 1349-0958

  13. 低温電子顕微鏡法による生体超分子の三次元構造解析

    米倉 功治, 眞木 さおり

    顕微鏡　42　(1)　29-34　2007/03

    Publisher: (公社)日本顕微鏡学会

    ISSN： 1349-0958

  14. 【生命秩序を担う生体超分子】巨大構造体の形成・機能発現における超分子相互作用 べん毛による細菌の泳ぎと方向転換の分子機構

    今田 勝巳, Samatey Fadel A., 松波 秀行, 長島 重広, 北尾 彰朗, 米倉 功治, 眞木 さおり, 難波 啓一

    蛋白質・核酸・酵素　50　(10)　1328-1334　2005/08

    Publisher: 共立出版(株)

    ISSN： 0039-9450

  15. 電子顕微鏡で解かれたべん毛ナノマシン

    米倉 功治, 眞木 さおり, 難波 啓一

    蛋白質・核酸・酵素　49　(4)　534-542　2004/03

    Publisher: 共立出版(株)

    ISSN： 0039-9450

  16. 低温電子顕微鏡によるリボソーム-RRF複合体の立体構造解析

    加藤 貴之, 米倉 功治, 松尾 瞳, 吉田 卓也, 大久保 忠恭, 難波 啓一, 小林 祐次

    生化学　76　(3)　298-298　2004/03

    Publisher: (公社)日本生化学会

    ISSN： 0037-1017

  17. 低温電子顕微鏡法によるバクテリアべん毛フィラメントの原子モデル (日本顕微鏡学会第48回シンポジウム 材料科学と生命科学のクロストーク--顕微解析の最前線) -- (生物系セッション1 電子顕微鏡による膜蛋白質の高分解能構造解析)

    米倉 功治, 眞木 さおり, 難波 啓一

    電子顕微鏡　38　3-6　2003

    Publisher: 日本顕微鏡学会

    ISSN： 0417-0326

  18. Na+駆動型べん毛モータのイオン透過蛋白PomABの大量精製

    渥美龍男, 小嶋勝, 薬師寿治, 本間道夫, 米倉功治, 上池伸徳, 大沢研二, 難波啓一

    日本生物物理学会年会講演予稿集　40th　2002

    ISSN： 0582-4052

  19. 細菌べん毛繊維の自己構築とらせんスイッチ機構 (特集 ここまで進んだ構造生物学--立体構造解析から生命現象を解き明かす)

    難波 啓一, 米倉 功治, 眞木 さおり

    細胞工学　20　(10)　1371-1379　2001/10

    Publisher: 秀潤社

    ISSN： 0287-3796

  20. 【新世紀における蛋白質科学の進展】蛋白質の構造・物性・進化 分子集合のメカニズム 細菌べん毛の分子構造から自己構築とサブオングストローム精度のスイッチ機構にせまる

    難波 啓一, 米倉 功治, 眞木 さおり, Samatey Fadel A., 今田 勝巳

    蛋白質・核酸・酵素　46　(11)　1568-1576　2001/08

    Publisher: 共立出版(株)

    ISSN： 0039-9450

  21. Structural mechanisms of self-assembly and polymorphic supercoiling of the bacterial flagellum (日本電子顕微鏡学会第46回シンポジウム 材料のナノ・生物のナノ) -- (共通セッション1 タンパクマシン--"7th International Symposium on Advanced physical Fields(APF-7)"との合同セッション)

    難波 啓一, Yonekura Koji, Maki-Yonekura Saori

    電子顕微鏡　36　61-64　2001

    Publisher: 日本電子顕微鏡学会

    ISSN： 0417-0326

  22. 正二十面体ウイルスのキャプシドの多形成 イネ萎縮ウイルスRDVの場合

    岩崎 憲治, Zhu Yafeng, 大村 敏博, 米倉 功治, 豊島 近, 村田 和義, 難波 啓一, Cheng Holland, 藤吉 好則

    生化学　72　(8)　762-762　2000/08

    Publisher: (公社)日本生化学会

    ISSN： 0037-1017

  23. イオンチャネル,ポンプの構造と機能 (構造生物学のフロンティア--シグナル伝達とDNAトランスアクション) -- (膜蛋白質)

    豊島 近, 米倉 功治

    蛋白質核酸酵素　44　(4)　557-565　1999/03

    Publisher: 共立出版

    ISSN： 0039-9450

  24. Axial projection map of bacterial flagellar FliF ring

    Suzuki H., Yonekura K., Murata K., Hirai K., Oosawa K., Namba K.

    Biophysics　38　(2)　S70　1998/09/07

    Publisher: The Biophysical Society of Japan General Incorporated Association

    ISSN： 0582-4052

  25. The structure of bacterial flagella and its cap protein, HAP2 complex

    Yonekura K., Maki S., Morgan D.G., DeRosier D.J., Vonderviszt F., Imada K., Namba K.

    Biophysics　38　(2)　S71　1998/09/07

    Publisher: The Biophysical Society of Japan General Incorporated Association

    ISSN： 0582-4052

  26. P型イオンポンプの立体構造

    米倉 功治, 豊島 近

    細胞工学　17　(8)　1270-1271　1998/08

    Publisher: (株)学研メディカル秀潤社

    ISSN： 0287-3796

  27. Transmembrane architecture of the calcium pump from sarcoplasmic reticulum by cryoelectron microscopy

    P Zhang, NM Green, C Toyoshima, K Yonekura, DL Stokes

    BIOPHYSICAL JOURNAL　74　(2)　A328-A328　1998/02

    ISSN： 0006-3495

  28. Three dimensional structure of photosystem II core complex

    MAYANAGI K., ISHIKAWA T., TOYOSHIMA C., YONEKURA K., TANI K., INOUE Y., NAKAZATO K.

    Biophysics　37　S165　1997/10

    Publisher: The Biophysical Society of Japan General Incorporated Association

    ISSN： 0582-4052

  29. 筋小胞体カルシウムATPaseの構造と機能

    米倉 功治, 豊島 近

    生物物理　37　(1)　310-314　1997/01

    Publisher: (一社)日本生物物理学会

    ISSN： 0582-4052

    eISSN： 1347-4219

Show all ︎Show first 5

Presentations 146

  1. 基調講演 Invited

    米倉 功治

    3DED/microED 講習会 〜電子回折で拓くナノ結晶構造解析の新時代〜 有機・無機・生体分子を対象に　2025/09/09

  2. 電子線三次元結晶構造解析とクライオ EM による化学特性の計測 Invited

    米倉 功治

    第53回構造活性相関シンポジウム　2025/09/05

  3. Measurement of chemical information using 3D ED and Cryo-EM International-presentation Invited

    Koji Yonekura

    The 75th Annual Meeting of the American Crystallographic Association　2025/07/22

  4. 低GC生物マイコプラズマ由来新規Cas9の探索

    上野 莉香子, 池内 健, 豊永 拓真, 濵口 祐, 米倉 功治

    公益社団法人日本顕微鏡学会 第81回学術講演会　2025/06/11

  5. CryoEMによって可視化したマイコプラズマ滑走型Rotary ATPaseの回転

    豊永拓真, 海原大輔, 濵口 祐, 米倉功治

    公益社団法人日本顕微鏡学会 第81回学術講演会　2025/06/09

  6. クライオEELS/EF-TEMによる凍結溶媒内バイオマテリアルの多元素マッピング観察

    海原大輔, 佐藤庸平, 濵口祐, 米倉功治

    公益社団法人日本顕微鏡学会 第81回学術講演会　2025/06/10

  7. Cryo-EM Analysis of Chained G1-ATPase, Motor for Mycoplasma Gliding International-presentation

    Takuma Toyonaga, Daisuke Unabara, Tasuku Hamaguchi, Makoto Miyata, Koji Yonekura

    Sixth Japan-Canada Microscopy Societies Joint Symposium 2025　2025/06/10

  8. クライオEMとEDによる化学情報の計測 Invited

    米倉 功治

    量子生命科学会第7回大会　2025/05/28

  9. Exploring chemical properties through 3D ED and Cryo-EM International-presentation Invited

    Koji Yonekura

    7th International Symposium on Diffraction Structural Biology (ISDSB2025).　2025/05/06

  10. クライオ電⼦顕微鏡、電⼦線三次元結晶構造解析による化学情報の測定 Invited

    化研セミナー：電⼦顕微鏡研究の進歩と未来　2025/04/15

  11. Visualizing hydrogen by 3D ED crystallography International-presentation

    Saori Maki-Yonekura, Koji Yonekura

    Japan Society for Promotion of Science (Alumni Club Seminar)　2025/02/13

  12. Exploring chemical properties through 3D ED and cryo-EM Invited

    Koji Yonekura

    Japan Society for Promotion of Science (Alumni Club Seminar)　2025/02/13

  13. Chirality Determination Using Qualitative Bijvoet Difference by 3D ED Analysis International-presentation

    Tianyu Liu, Daisuke Morikawa, Kurokawa Hirofumi, Shun Narai, Yusuke Seto, Koji Yonekura, Kenji Tsuda

    The 13th Asia Pacific Microscopy Congress 2025 (APMC13)　2025/02/05

  14. Cryo-EM Structure of PSI-LHCI from a Red alga Cyanidium caldarium International-presentation

    Koji Kato, Tasuku Hamaguchi, Minoru Kumazawa, Yoshiki Nakajima, Kentaro Ifuku, Yuu Hirose, Keisuke Kawakami, Koji Yonekura, Ryo Nagao, Jian-Ren Shen

    The 13th Asia Pacific Microscopy Congress 2025 (APMC13)　2025/02/04

  15. Exploring chemical properties through 3D ED and cryo-EM International-presentation Invited

    Koji Yonekura, Saori Maki-Yonekura

    The 13th Asia Pacific Microscopy Congress 2025 (APMC13)　2025/02/05

  16. マイコプラズマ滑走運動を駆動する新奇回転分子モーターのCryoEM構造

    豊永拓真, 濵口 祐, 宮田真人, 米倉功治

    第24回東北大学多元物質科学研究所研究発表会　2024/12/12

  17. クライオFIB-SEMおよびクライオTEMを利用した納豆芽胞の可視化

    丹羽瑠美, 濵口祐, 米倉功治

    第24回東北大学多元物質科学研究所研究発表会　2024/12/12

  18. クライオEELS/EF-TEMによる凍結溶媒内ソフトマテリアルの元素マッピング観察

    海原大輔, 佐藤庸平, 濵口祐, 米倉功治

    第24回東北大学多元物質科学研究所研究発表会　2024/12/12

  19. Chirality identification using qualitative Bijvoet difference by 3D ED analysis

    T. Liu, D. Morikawa, H. Kurokawa, S. Narai, Y. Seto, K. Yonekura, K. Tsuda

    2024/11/03

  20. クライオ電子顕微鏡による化学構造解析への挑戦 Invited

    米倉 功治

    CSJ化学フェスタ2024　2024/10/24

  21. Structure of the far-red light utilizing photosystem II interacting with chlorophyll-binding protein (CBP) from Acaryochloris marina at 2.4 Å resolution International-presentation Invited

    Keisuke Kawakami, Saori Maki-Yonekura, Kyoko Shinzawa-Itoh, Natsuko Inoue-Kashino, Shigeru Itoh, Kentaro Ifuku, Koji Yonekura, Yasuhiro Kashino

    12th International Conference: Photosynthesis and Hydrogen Energy Research for Sustainability　2024/10/15

  22. Structure of the far-red light utilizing photosystem I of Acaryochloris marina International-presentation

    Keisuke Kawakami, Saori Maki-Yonekura, Kyoko Shinzawa-Itoh, Natsuko Inoue-Kashino, Shigeru Itoh, Kentaro Ifuku, Koji Yonekura, Yasuhiro Kashino

    2nd Asia-Oceania International Congress on Photosynthesis (AOICP),　2024/09/18

  23. Cryo-EM structure of PSI-LHCI from a red alga Cyanidium caldarium International-presentation

    Koji Kato, Tasuku Hamaguchi, Minoru Kumazawa, Yoshiki Nakajima, Kentaro Ifuku, Yuu Hirose, Keisuke Kawakami, Koji Yonekura, Ryo Nagao, Jian-Ren Shen

    2nd Asia-Oceania International Congress on Photosynthesis (AOICP)　2024/09/18

  24. Measurement of charges and chemical bonding using CRYO ARM International-presentation Invited

    Koji Yonekura

    International CRYO ARM user meeting　2024/09/11

  25. フェムト秒分光を用いた遠赤色光利用可能な光化学系IIにおける電子・エネルギー伝達機構の解明

    小澄大輔, 川上恵典, 米倉功治, 新澤(伊藤)恭子, 井上(菓子野, 名津子, 菓子野康浩

    2024年光化学討論会　2024/09/03

  26. Analysis of chemical properties using 3D ED and other techniques International-presentation Invited

    Koji Yonekura

    34th European Crystallographic Meeting (ECM)　2024/08/30

  27. Approaches to quantum chemistry using microcrystals: electron and XFEL crystallography International-presentation

    Kiyofumi Takaba, Saori Maki-Yonekura, Koji Yonekura

    34th European Crystallographic Meeting (ECM)　2024/08/27

  28. Applications of cryo-EM and XFEL crystallography International-presentation

    Koji Yonekura, Saori Maki-Yonekura

    DIFFRACTION METHODS IN STRUCTURAL BIOLOGY 2024　2024/07/25

  29. Measurement of charges and chemical bonding in Cryo-EM SPA International-presentation Invited

    Saori Maki-Yonekura, Keisuke Kawakami, Koji Yonekura

    DIFFRACTION METHODS IN STRUCTURAL BIOLOGY 2024　2024/07/25

  30. XFEL and Electron Crystallography of Organic Compounds International-presentation Invited

    Koji Yonekura

    The American Crystallographic Association's 74th Annual Meeting　2024/07/10

  31. 光合成超複合体の調製とその特性評価 Invited

    川上 恵典, 小澄 大輔, 眞木 さおり, 米倉 功治

    第31回光合成セミナー：反応中心と色素系の多様性　2024/06/30

  32. クライオ電顕の単粒子解析, 電子回折解析とAI測定 Invited

    米倉功治

    日本表面真空学会真空技術部会2024年 6月研究例会「AIによる表面科学・真空技術の新展開」　2024/06/21

  33. Understanding Crystal Surface Molecular Arrangement:Integration of Optical/Scanning Electron Microscopy and 3D ED/MicroED International-presentation Invited

    Shun Narai, Koji Yonekura

    Fifth Japan-Canada Microscopy Societies Joint Symposium 2024　2024/06/04

  34. クライオEELS/EF-TEMによる凍結溶媒内ソフトマテリアルの元素マッピング観察

    海原 大輔, 佐藤 庸平, 濵口 祐, 米倉 功治

    日本顕微鏡学会 第80回学術講演会　2024/06/04

  35. 溶液中の粒子配向を改善した高精度単粒子解析

    岡 圭吾, 濵口 祐, 海原 大輔, 米倉 功治

    日本顕微鏡学会 第80回学術講演会　2024/06/03

  36. クライオ電子顕微鏡による生体分子構造解析技術の開発と応用

    米倉 功治

    日本顕微鏡学会 第80回学術講演会　2024/06/04

  37. Measurement of charges and chemical bonding in cryo-EM SPA and 3D ED structures International-presentation Invited

    Koji Yonekura

    5th International Symposium on Cryo-3D Image Analysis 2024　2024/03/15

  38. プロジェクトの概要説明

    米倉 功治

    JST未来社会創造事業『超原子座標構造の可視化による創薬の革新』本格研究キックオフ公開シンポジウム　2024/03/08

  39. Mixing-Triggered Control of HigherMolecular Ordering in Layered Organic Semiconductors through Alkyl Chain Interdigitation International-presentation

    K. Nikaido, S. Inoue, S. Tsuzuki, R. Kumai, K. Takaba, S. Maki-Yonekura, K. Yonekura, T. Hasegawa

    MRM2023/IUMRS-ICA2023　2023/12

  40. Investigation and application of solvent effects in high-resolution single-particle cryogenic-electron microscopy International-presentation

    Keigo Oka, Tasuku Hamaguchi, Daisuke Unabara, Koji Yonekura

    2023 Joint Symposium, National Taipei of University of Technologies and Tohoku University　2023/12/11

  41. Measurement of charges and chemical bonding through single-particle cryo-EM and ED International-presentation Invited

    Koji Yonekura

    2023 Joint Symposium　2023/12/11

  42. クライオ EM，ED と XFEL による電荷，化学結合の計測 Invited

    米倉 功治

    R022 量⼦構造⽣物学委員会 第12回研究会　2023/12/05

  43. 超原子座標構造の計測 Invited

    米倉 功治

    生理研研究会2023「クライオ電子顕微鏡とその周辺」　2023/11/28

  44. Applications and limitations of electron 3D crystallography International-presentation Invited

    Koji Yonekura

    CRYOZ 2023　2023/11/23

  45. Measurement of charge and chemical bonding through cryo-EM and ED Invited

    Koji Yonekura

    Frontiers in Microscopy & Microanalysis Seminars　2023/11/22

  46. Cryo-EM observation of wide range of soft-materials International-presentation Invited

    Tasuku Hamaguchi, Keisuke Kawakami, Daisuke Unabara, Koji Yonekura

    2023/11/14

  47. Cryo-EM, ED analyses and AI data collection Invited

    Koji Yonekura

    2023/11/12

  48. 多様な液体材料観察を可能とするクライオEM試料作製法の検討とトモグラフィー解析

    海原大輔, 濱口祐, 米倉功治

    日本顕微鏡学会 第66回シンポジウム　2023/11/12

  49. Measurement of charges and chemical bonding in single-particle cryo-EM and 3 D ED structures Invited

    Koji Yonekura

    2023/11/02

  50. クライオEMと3D EDによる電荷、化学結合の計測 Invited

    米倉 功治

    令和5年(2023年)度 日本結晶学会年会および会員総会　2023/10/27

  51. フィコビリソームにおける エネルギー伝達経路

    城戸功史朗, 野地 智康, 川上恵典, 斉藤 圭亮, 石北 央

    第13回CSJ化学フェスタ2023　2023/10/19

  52. クライオTEMによる解析技術の進展 - クライオTEMのマテリアルへの応用展開 -

    米倉 功治

    東北大学 多元物質科学研究所「次世代電子顕微鏡技術共同研究部門」成果報告会　2023/10/13

  53. 層状有機半導体pTol-BTBT-Cn混合系の電子回折による結晶構造解析：層間アルキル噛み合わせ効果と高秩序相発現

    Kiyoshi Nikaido, Satoru Inoue, Seiji Tsuzuki, Reiji Kumai, Kiyofumi Takaba, Saori Maki-Yonekura, Koji Yonekura, Tatsuo Hasegawa

    2023/09/22

  54. X 自由電子レーザーによるフェムト秒光電子反応過程の第一原理分子動力学法に基づく解釈

    高 章磊, 高場 圭章, 米倉 功治, 岸本 直樹

    第17回分子科学討論会2023大阪　2023/09/15

  55. 遠赤色光利用可能な光化学系IIの光捕集機能

    Joris Vasco, 浜 勇二朗, 板東(魚谷) 未季, 新澤(伊藤) 恭子, 井上(菓子野, 名津子, 菓子野 庸浩, 川上 恵典, 米倉 功治, 小澄 大輔

    第17回分子科学討論会2023大阪　2023/09/13

  56. アキラルCo(Ⅱ)フタロシアニンで構成された螺旋超分子のMC-AFMによる電流測定

    相澤 洋紀, 佐藤 拓朗, 米倉 功治, 米倉‐眞木 沙織, 濱口 祐, 高場 圭章, 湊 丈俊, 山本 浩史

    第17回分子科学討論会2023大阪　2023/09/13

  57. Cryo-electron microscopy for chemical analysis International-presentation Invited

    Koji Yonekura

    International Symposium for the 80th Anniversary of the Tohoku Branch of the Chemical Society of Japan (2023 Joint Meeting of the Tohoku Area Chemistry Societies)　2023/09/08

  58. A study on structures of N-methyl peptides toward a bottom-up nanostructure design Invited

    MORIMOTO Jumpei, SHIRATORI Yota, YOKOMINE Marin, UEDA Takumi, NAKAMURO Takayuki, TAKABA Kiyofumi, MAKI-YONEKURA Saori, UMEZAWA Koji, MIYANISHI Koichiro, FUKUDA Yasuhiro, WATANABE Takumu, MIZUKAMI Wataru, TAKEUCHI Koh, YONEKURA Koji NAKAMURA, Eiichi, SANDO Shinsuke

    2023/09

  59. Photosynthetic functions of photosystem II substituted to iodine

    Yujiro Hama, Mizuki Noai, Miki Bando-Uotani, Keisuke Kawakami, Koji Yonekura, Nobuo Kamiya, Daisuke Kosumi

    2023/09

    More details Close

    Photosystem II is an essential protein complex driving a water splitting reaction by sunlight in oxygenic photosynthesis. Photosystem II is consisting of a number of proteins and cofactors, and it has been suggested that chloride ion plays important roles in water-splitting reaction. In this study, we performed on femtosecond pump-probe spectroscopic measurements on PSII substituted to iodine to clarify roles of iodine in photosynthetic function.

  60. Complementary use of electron and X-ray microcrystallography to reveal quantum structural information of organic molecules International-presentation Invited

    K. Takaba, S. Maki-Yonekura, I. Inoue, K. Tono, M. Yabashi, K. Yonekura

    26TH CONGRESS AND GENERAL ASSEMBLY OF THE INTERNATIONAL UNION OF CRYSTALLOGRAPHY(IUCr2023)　2023/08/25

  61. Cryo-EM observation of γ-polyglutamic acid hydrogels from Bacillus subtilis of Firmicutes International-presentation

    Tasuku Hamaguchi, Daisuke Unabara, Koji Yonekura

    24th Biennial Congress of the International Organization for Mycoplasmology　2023/07/17

  62. Structure of the far-red light utilizing photosystem I of Acaryochloris marina International-presentation Invited

    Keisuke Kawakami, Tasuku Hamaguchi, Kyoko Shinzawa-Itoh, Natsuko, Inoue-Kashino, Shigeru Itoh, Kentaro Ifuku, Eiki Yamashita, Kou Maeda, Koji Yonekura, Yasuhiro Kashino

    11th International Conference Photosynthesis and Hydrogen Energy Research for Sustainability – 2023»　2023/07

  63. 3D EDから得られる構造情報とその利用 Invited

    高場圭章, 眞木さおり, 米倉功治

    日本顕微鏡学会第79回学術講演会　2023/06/26

  64. Cryo-EM observations for proteins and soft-materials International-presentation Invited

    Tasuku Hamaguchi, Keisuke Kawakami, Daisuke Unabara, Koji Yonekrura

    International symposium for young scientist in state-of-the-art microscopy, The 79th Annual Meeting of The Japanese Society of Microscopy　2023/06/25

  65. Acaryochloris marina由来光化学系II複合体の構造

    川上 恵典, 新澤(伊藤)恭子, 眞木(米倉)さおり, 井上(菓子野, 名津子, 伊福 健太郎, 伊藤 繁, 米倉 功治, 菓子野 康浩

    第30回「光合成セミナー2023」　2023/06/24

    More details Close

    光合成に関して、物理学、化学、生物学を融合した討論を行う。光合成の進化、物質変換、人工光合成などについても討論する。第一線の研究者に最新のトピックを解説していただくとともに、参加者の口頭・ポスター発表を行う。

  66. Photosynthetic functions of carotenoids in photosystem I from cyanobacteria, as revealed by ultrafast spectroscopy

    Taiki Nohara, Miki Bando-Uotani, Hiroki Serikawa, Keisuke Kawakami, Koji Yonekura, Nobuo Kamiya, Daisuke Kosumi

    The 19th International Symposium on Carotenoids　2023/06

  67. Excitation energy transfer dynamics of carotenoids in the far-red light utilizing photosystem II from Acaryochloris marina

    Miki Bando-Uotani, Kyoko Shinzawa-Itoh, Natsuko Inoue-Kashino, Yasuhiro Kashino, Keisuke Kawakami, Koji Yonekura, Daisuke Kosumi

    The 19th International Symposium on Carotenoids　2023/06

  68. クライオEM の分⼦像と電⼦回折測定のAI 制御「ソフトマテリアルの顕微鏡解析でAIをどう活用できるか」, Invited

    米倉 功治

    日本顕微鏡学会 ソフトマテリアル分科会 2022 年度 第2回講演会　2023/02/18

  69. Collaboration of photons and electrons: visualization of reacting quantum structures International-presentation Invited

    Kiyofumi Takaba

    The 12th Ringberg Workshop on Science with FEL　2023/02/07

  70. 多元物質科学研究所におけるクライオ電子顕微鏡を用いた取り組み（利用方法から観察事例まで） Invited

    濵口祐

    生産性に効く！最先端ナノ計測評価技術, 東北大学多元物質科学研究所 イノベーション・エクスチェンジ2022　2023/01/18

  71. 立体構造から見る光合成蛋白質の類似性と多様性 Invited

    川上 恵典

    藍藻の分子生物学2022　2022/12/09

  72. SPring-8/SACLA, 電子顕微鏡を使ったタンパク質の研究

    川上 恵典

    SPring-8/SACLAの高校生見学対応　2022/12/08

  73. MicroEDとSPring-8/SACLAから得られる結晶構造とその利用 Invited

    高場圭章

    CBI研究機構 量子構造生命科学研究所 中性子産業利用推進協議会 生物・生体材料研究会 合同シンポジウム「MicroEDの現状と未来」　2022/12/06

  74. クライオEM、3D EDによる高度解析 Invited

    米倉 功治

    未踏分析技術イノベーションウェビナー ～日本発未踏技術を世界の産業活性にテイクオフさせる必見企画～　2022/12/06

  75. 光合成タンパク質における超高速エネルギー・電子伝達ダイナミクス Invited

    小澄 大輔, 板東(魚谷) 未希, 川上 恵典, 米倉 功治, 神谷 信夫

    電子スピンサイエンス学会2022　2022/12/04

  76. Core and rod structures of a thermophilic cyanobacterial light-harvesting phycobilisome International-presentation

    Keisuke Kawakami, Tasuku Hamaguchi, Yuu Hirose, Daisuke Kosumi, Makoto Miyata, Nobuo Kamiya, Koji Yonekura

    AsBIC10 2022　2022/11/30

  77. Analysis of Japanese sake using synchrotron light and cryo-TEM International-presentation Invited

    Tasuku Hamaguchi

    Switzerland - Japan Workshop for Synchrotron Light and Wine/Sake　2022/11/25

  78. 理研・東北大の施設報告 Invited

    濱口 祐

    AMED/BINDS第1回クライオ電子顕微鏡施設技術交流会　2022/11/21

  79. Challenges on microED, for atomic charge determination Invited

    2022/11/21

  80. Structure details and properties of small organic molecules to proteins revealed by single particle cryo-EM, 3D ED and XFEL International-presentation Invited

    Saori Maki-Yonekura

    The Asia Oceania International Conference on Synchrotron Radiation Instruments 2022(AO-SRI2022)　2022/11/10

  81. Two-way application of cryo-EM to high-resolution single particle analysis and 3D ED International-presentation Invited

    2022/11/11

  82. 3D EDのためのFIBSEM利用の現状 Invited

    高場圭章

    第13回藍藻の分生成物学2022日本顕微鏡学会 生体解析分科会研究会「バイオ向けクライオ透過電子顕微鏡の多様性の最前線３」　2022/11/02

  83. Organic molecular crystallography by 3D ED and XFEL International-presentation Invited

    Saori Maki-Yonekura

    AsCA 2022　2022/10/31

  84. 高精度クライオEMによる構造解析 Invited

    濱口 祐

    生理研研究会「新世代のクライオ電子顕微鏡」　2022/11/01

  85. Two-way Application of Single Particle Cryo-EM and 3D ED for High-resolution Structure Analyses International-presentation Invited

    Koji Yonekura

    AsCA 2022　2022/10/31

  86. 電子顕微鏡と放射光の相補による微小結晶構造解析と材料分子化学への応用 Invited

    高場 圭章

    CREST「革新材料開発」領域会議（第 9 回）　2022/10/30

  87. CYPを対象としたCryo-EM Invited

    米倉 功治, 濵口 祐, 高場 圭章, 川上 恵典, 眞木 さおり

    CBI学会2022年大会　2022/10/25

  88. クライオ電子顕微鏡による有機分子観察について Invited

    米倉 功治

    東北大学-DOWA 技術者交流　2022/10/12

  89. 構造解析を行うための、光合成蛋白質試料の純化とその評価

    川上 恵典

    第2回光合成タンパク質勉強会　2022/10/08

  90. High-precision structural analysis by cyro-electron microscopy

    Tasuku Hamaguchi, Keisuke Kawakami, Saori Maki-Yonekura, Koji Yonekura

    2022/09/30

  91. 立体構造解析に基づいた光合成タンパク質複合体内のカロテノイド同定

    川上 恵典

    第34回カロテノイド研究談話会　2022/09/17

  92. 光合成シアノバクテリアThermosynechococcus vulcanus由来光化学系IIに結合するβ-カロテン励起状態ダイナミクスの励起波長依存性

    板東(魚谷) 未希, 木田 雅俊, 川上 恵典, 米倉 功治, 神谷 信夫, 小澄 大輔

    第34回カロテノイド研究談話会　2022/09/17

  93. 好熱性シアノバクテリア由来フィコシアニン三量体におけるサイトエネルギーとエネルギー伝達

    木田 雅俊, 川上 恵典, 米倉 功治, 神谷 信夫, 小澄 大輔

    2022年光化学討論会　2022/09/15

  94. 超高速分光を用いたシアノバクテリア光化学系Iにおける光捕集機構解明

    小澄 大輔, 板東(魚谷) 未希, 木田 雅俊, 野原 大輝, 川上 恵典, 米倉 功治, 神谷 信夫

    2022年光化学討論会　2022/09/15

  95. 創薬

    濵口 祐

    ソフトマテリアル研究拠点会議　2022/08/10

  96. 微小結晶3D解析（有機半導体/薬剤/材料）2

    眞木 さおり

    ソフトマテリアル研究拠点会議　2022/08/10

  97. 放射光で解析困難なナノ有機結晶の構造解析-クライオ電顕＋3D ED解析による構造解析-

    黒河 博文

    ソフトマテリアル研究拠点会議　2022/08/10

  98. Synchrotron and electron crystallography to evaluate noncovalent interactions in soft matters International-presentation

    Kiyofumi Takaba, Saori Maki-Yonekura, Ichiro Inoue, Kensuke Tono, Yang Tai, Ryota Takeda, Kazuki Takeda, Tetsuya Ishikawa, Makina Yabashi, Koji Yonekura

    The second International Conference on Noncovalent Interactions 2021-22　2022/07/19

  99. クライオEMデータのAI測定 ～医薬品開発への応用～ Invited

    米倉 功治

    第4回再生医療EXPO東京　2022/07/14

  100. シアノバクテリア光化学系 I におけるレッドクロロフィルの役割

    小澄大輔, 板東(魚谷)未季, 木田雅俊, 野原大暉, 川上恵典, 米倉功治, 神谷信夫

    第29回「光合成セミナー2022」　2022/06/25

  101. シアノバクテリア由来フィコシアニン三量体の光捕集機能 1~フィコシアニン三量体中のサイトエネルギーとエネルギー伝達~

    木田雅俊, 川上恵典, 米倉功治, 神谷信夫, 小澄大輔

    第29回「光合成セミナー2022」　2022/06/25

  102. 100 フェムト秒ポンプ・プローブ分光測定を用いた光化学系Ⅱにおけるエネルギー・電子伝達の温度依存性の観測

    板東(魚谷)未季, 木田雅俊, 川上恵典, 米倉功治, 神谷信夫, 小澄 大輔

    第29回 光合成セミナー2022　2022/06/25

  103. 好熱性シアノバクテリア由来フィコビリソームコアとフィコシアニンロッドのクライオ電子 顕微鏡解析

    川上恵典, 浜口 祐, 広瀬 侑, 小澄大輔, 宮田真人, 神谷信夫, 米倉功治

    第29回 光合成セミナー2022　2022/06/25

  104. クロロフィル d を持つ A. marina PSI の遠赤色光の捕集機構：色素交換仮想実験による T. elongatus PSI との機能的差異

    木村明洋, 鬼頭宏任, 青田俊道, 浜口祐, 米倉功治, 川上恵典, 新沢-伊藤 恭子, 井上-菓子野名津子, 伊福健太郎, 菓子野康浩, 山下栄樹, 伊藤 繁

    第29回 光合成セミナー2022　2022/06/25

  105. シアノバクテリア由来フィコシアニン三量体における光捕集機能2~フィコシアニン三量体中の量子コヒーレンス~

    小澄 大輔, 木田 雅俊, 川上 恵典, 米倉 功治, 神谷 信夫

    第29回「光合成セミナー2022」　2022/06/25

  106. Hydrogen properties and charges in crystal and single particle cryo-EM structures International-presentation Invited

    Saori Maki-Yonekura

    The 2022 Gordon Research Conference(GRC)　2022/06/19

  107. 量子アニーリングで期待されるクライオEM解析と構造インフォマティクスへの新展開 Invited

    米倉功治

    ソフトマテリアル研究拠点 第2回シンポジウム　2022/03/07

  108. クライオEM とED による高精度解析ー水素の可視化とその先へ Invited

    米倉功治

    ＣＢＩ研究機構 量子構造生命科学研究所 中性子産業利用推進協議会 生物・生体材料研究会 合同シンポジウム　2022/03/04

  109. Sub-Å resolution SACLA-SFX for organic chemicals

    Takaba K, Maki-Yonekura S, Yonekura K

    SACLA Users’ Meeting 2022 Breakout Sessions　2022/03/02

  110. クライオ電顕の最前線 — 水素の可視化とその先の解析— Invited

    米倉功治

    コロイド先端技術講座2021 先端バイオ計測技術・研究から学び，コロイド界面化学のあらたな見方を探る　2022/02/08

  111. クライオEMとXFELによる構造解析の展開 -- 水素の可視化とその先へ -- Invited

    米倉功治

    第７回CYP研究会　2021/12/22

  112. High-resolution and high-precision cryo-ED and EM International-presentation Invited

    Yonekura K, Maki-Yonekura S, Takaba K, Hamaguchi T, Naitow H, Kawakami K

    Biological Structural Dynamics by Ultrafast X-ray and 4D Electron Microscopy, Pacifichem 2021　2021/12/16

  113. クライオ電子顕微鏡と放射光の相補的解析-- 水素の可視化とその先の解析 -- Invited

    米倉功治

    次世代放射光からつながる広がる可視化ツール, 東北大学多元物質科学研究所 イノベーション・エクスチェンジ2021　2021/12/10

  114. Two-way application of cryo-EM to high-resolution and high-precision SPA and EX--- Visualization of hydrogen and beyond International-presentation Invited

    Yonekura K

    Asia-Pacific Cryo-EM Symposium　2021/12/07

  115. AI 制御による自動電子線三次元結晶構造解析 Invited

    米倉功治

    「メソ材料精密制御」ワークショップ　2021/12/02

  116. Hydrogen properties revealed by cryo-EM and ED International-presentation Invited

    Yonekura K

    MIRAI 2.0 Materials Science Workshop　2021/12/01

  117. Two-way use of cryo-EM for SPA and 3D ED -- Revealing hydrogen and beyond International-presentation Invited

    Yonekura K

    Improve your Cryo-EM results, virtual round table, Quantifoil Inc.　2021/09/15

  118. クライオEMと EDの進展 -- 水素の可視化とその先 – Invited

    米倉功治

    東京大学大学院工学系研究科 総合研究機構 第５回「次世代電子顕微鏡法」講演会　2021/08/26

  119. The development of 3D ED on studying structure and charge state of protein microcrystals International-presentation Invited

    Yonekura K, Maki-Yonekura S, Takaba K, Hamaguchi T, Naitow H, Kawakami K

    25th IUCr Congress Satellite School on Electron Crystallography　2021/08/11

  120. Complementary use of high-resolution and high-precision cryo-ED and EM International-presentation Invited

    Yonekura K, Maki-Yonekura S, Takaba K, Hamaguchi T, Naitow H, Kawakami K

    Microscopy and Microanalysis (M&M) 2021　2021/08/02

  121. クライオEM, EDによる高分解能高精度構造解析 Invited

    米倉功治

    日本顕微鏡学会第77回学術講演会　2021/06/14

  122. クライオ電顕を用いたソフトマテリアル解析 Invited

    米倉功治

    ソフトマテリアル研究拠点第１回シンポジウム 東北大学(Online) 2021年3月31日　2021/03/31

  123. クライオ EM, ED による高精度高分解能構造析-- タンパク質から有機低中分子への応用 Invited

    米倉功治

    日本学術振興会「分子系の複合電子機能第181委員会」第33回研究会　2021/02/26

  124. Cold-field emission電子ビームによる高分解能リモート単粒子解析 Invited

    米倉功治

    生理研研究会「クライオ電子顕微鏡によるタンパク質の高分解能単粒子構造解析」～ コロナ禍にできること、すべきこと～　2020/11/10

  125. クライオ電顕技術進化と次世代先端計測手法としての展望 Invited

    米倉功治

    東北大学多元研ソフトマテリアル研究拠点キックオフ・セミナー　2020/08/04

  126. Cryo-ED and EM for higher-resolution and higher-precision structure analysis International-presentation Invited

    Yonekura, K, Maki-Yonekura, S. Hamaguchi, T, Naitow, H, Kiyofumi, T

    Japan-Canada Joint Symposium 2020, Development and applications of the methods combining interference/phase measurement techniques and analytic techniques, Japanese Society of Microscopy　2020/05/26

  127. Cryo-ED and EM for higher-resolution and higher-precision structure analysis International-presentation Invited

    Yonekura K

    Three Way Meeting at ESRF, Grenoble, France　2020/02/12

  128. Cryo-ED and EM for Higher-Resolution and Higher-Precision Structures International-presentation Invited

    Yonekura K, Maki-Yonekura S, Hamaguchi T, Naitow H, Takaba K

    12th Asia-Pacific Microscopy Conference (APMC2020) Hyderabad International Convention Center, Hyderabad, India　2020/02/04

  129. クライオ電子顕微鏡と放射光による構造研究 Invited

    米倉功治

    FSBL第2期発足記念式典・特別シンポジウム　2020/01/07

  130. クライオED, EMによる高分解能高精度解析－電子線三次元結晶解析と単粒子－ Invited

    米倉功治

    第12回 TEMユーザーズミーティング　2019/12/20

  131. クライオEM, EDによる高分解能高精度構造解析 Invited

    米倉功治

    国立国際医療研究センター, 研究所難治性疾患研究部セミナー　2019/11/21

  132. Cryo-EM and cryo-ED for higher-resolution and higher-throughput structural studies International-presentation Invited

    Yonekura K

    Luncheon seminar, 6th International Symposium on Diffraction Structural Biology　2019/10/18

  133. クライオ電子顕微鏡の材料科学研究への展開 Invited

    米倉功治

    JST CREST「実験・計算・データ科学融合による塗布型電子材料の開発 2019 第３回 チーム会議　2019/10/16

  134. クライオ電子顕微鏡のイメージングと回折による構造研究 Invited

    米倉功治

    第2回量子線イメージング研究会　2019/09/25

  135. 新型クライオ電子顕微鏡による高分解能構造解析 Invited

    米倉功治

    JASIS 2019 LSZ　2019/09/06

  136. クライオEM, EDによる高分解能高精度構造解析 Invited

    米倉功治

    JASIS 2019 コンファレンス 「SPring-8 / SACLA」　2019/09/05

  137. 電子分光三次元結晶構造解析のための新型クライオEMシステム Invited

    米倉功治, 眞木さおり

    日本顕微鏡学会第75回学術講演会 シンポジウム「クライオ電顕トモグラフィーが拓く未来生命科学」　2019/06/19

  138. Higher-resolution single particle analysis by a new cryo-EM system International-presentation Invited

    Yonekura K

    A Symposium, “Accelerating development of cryo-EM technology”, JSM 2019　2019/06/17

  139. A new cryo-EM system for higher-resolution single particle analysis and electron 3D crystallography International-presentation Invited

    Yonekura K

    3DEM, Gordon Research Conference　2019/06/10

  140. A new cryo-EM system for higher-resolution single particle analysis and electron 3D crystallography International-presentation Invited

    Yonekura K

    RSC Cryo-Electron Microscopy Symposium　2019/03/28

  141. 300kV CFEG cryo-EMによる電子線三次元結晶構造解析と高分解能単粒子解析 Invited

    米倉功治

    東京大学 第1257回生物科学セミナー　2019/03/07

  142. クライオEM, EDによる構造生物学 Invited

    米倉功治

    RSCミニシンポジウム　2019/02/08

  143. クライオEMと放射光による構造生物研究 Invited

    米倉功治

    第32回日本放射光学会年会・放射光科学合同シンポジウム　2019/01/10

  144. クライオ電子顕微鏡を用いた光合成による光エネルギー捕集機構の解明 Invited

    川上 恵典

    生産性に効く！最先端ナノ計測評価技術, 東北大学多元物質科学研究所 イノベーション・エクスチェンジ2022　2023/01/18

  145. 理研初号機と東北大多元研IIのリモート運用 Invited

    米倉 功治

    AMED/BINDS第1回クライオ電子顕微鏡施設技術交流会　2022/11/22

  146. クライオEMとeXによる高精度解析— 水素の可視化とその先へ Invited

    米倉 功治

    理研-JEOL連携センター講演会（研究成果報告会）　2022/10/19

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Research Projects 7

  1. Membrane potential-loaded structure of voltage-gated channels: Development of membrane potential-loaded synthetic membrane and single-particle structural analysis

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Challenging Research (Pioneering)

    Institution: University of Fukui

    2024/06/28 - 2027/03/31

  2. 超原子座標構造の可視化による創薬の革新

    米倉 功治

    Offer Organization: 科学技術振興機構

    System: 戦略的な研究開発の推進 未来社会創造事業 探索加速型

    Institution: 国立研究開発法人理化学研究所

    2023 - 2027

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    クライオ電子顕微鏡の先端技術開発を中心に据え、X線自由電子レーザー(XFEL)も用いることで、多様かつ微量な有機化合物、タンパク質などの試料から、高い時空間分解能とスピード解析を両立の上、これまでの計測限界を突破することを目指す。これにより、電荷分布、電子構造、化学結合の極性、官能基のプロトン化、電子の動き等“見えなかった”物性・現象、いわゆる“超原子座標構造”を解明する。まず、新規感染症や難病の治療に役立つ創薬への応用を進め、さらに、この技術の高い汎用性を活かし、新材料開発、エネルギー、環境、生命科学などより広い分野への応用も促進する。また、研究を通して次世代クライオ電顕を開発、世界シェアの拡大と解析拠点の構築にも繋げたい。以上のように、本可視化技術は共通基盤技術として、多くの研究開発現場における生産性向上に貢献することが期待される。

  3. Structural study of ion-driven energizers by cryo-EM and X-ray crystallography

    Koji Yonekura, Maki Saori

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Institute of Physical and Chemical Research

    2016/04/01 - 2019/03/31

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    Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. We present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation / inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

  4. Structural study of ExbB-ExbD-TonB that is responsible for energy transduction of proton motive force in bacteria

    Maki-Yonekura Saori

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (C)

    Institution: Institute of Physical and Chemical Research

    2015/04/01 - 2018/03/31

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    Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. We report the structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

  5. Visualization of Coulomb potential by electron crystallography

    YONEKURA Koji, MAKI Saori, TOYOSHIMA Chikashi

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Challenging Exploratory Research

    Institution: The Institute of Physical and Chemical Research

    2012/04/01 - 2015/03/31

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    Electron crystallography has the potential to analyze crystals of membrane proteins and macromolecular complexes too small or too thin for X-ray crystallography, as electrons are scattered 4 - 5 orders of magnitude more strongly than X-rays. Electron crystallography yields Coulomb potential maps, rather than electron density maps as X-rays do, providing information on charged states of amino-acids and metals. We presented such Coulomb potential maps at 3.4 and 3.2 Angstrom resolution, respectively, of calcium-ATPase and catalase obtained from crystals of just a few layers thick. These maps demonstrate that it is indeed possible to build atomic models from such crystals and charge information is included, often critical in understanding protein function.

  6. Structure analysis of S. cerevisiae Rap1-telomeric DNA complex by electron microscopy

    MAKI Saori, YONEKURA Koji, SAKO Yasushi, HIROSHIMA Michio, ELIZABETH H. Blackburn, TANYA L. Williams

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (C)

    Institution: The Institute of Physical and Chemical Research

    2008 - 2012

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    Telomeres are specialized nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes, stabilizing them and protecting their integrity. In budding yeast major telomere binding protein Rap1 controls telomere length. We prepared Rap1 and GFP fusion Rap1, and analyzed their structures by electron microscopy. Electron tomography and high resolution fluorescence microscopy revealed three-dimensional structures of unique filaments made of Rap1.

  7. Structural study of the torque-generating unit in the bacterial flagellar motor

    YONEKURA Koji, MAKI-YONEKURA Saori

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: The Institute of Physical and Chemical Research

    2008 - 2012

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    The bacterial flagellum is a rotary motor powered by the electrochemical potential of specific ions across the cytoplasmic membrane. There is little information about its working mechanism of this biological motor, the information which could be useful for biological science and nano-technology. In Vibrio, an inner membrane complex, PomAB, converts Na+ flux into rotation of the flagellum. We have been studying the structure of PomAB by X-ray crystallography and cryo-electron microscopy to elucidate the working mechanism of this energy conversion system.

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