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Improving particle behaviors for cryo-EM analysis by PEGylation

Research Project

Project/Area Number 21K19207
Research Category

Grant-in-Aid for Challenging Research (Exploratory)

Allocation TypeMulti-year Fund
Review Section Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
Research InstitutionThe University of Tokyo

Principal Investigator

Umeharu Ohto  東京大学, 大学院薬学系研究科(薬学部), 准教授 (90451856)

Project Period (FY) 2021-07-09 – 2023-03-31
Project Status Completed (Fiscal Year 2022)
Budget Amount *help
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2022: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Fiscal Year 2021: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Keywordsクライオ電子顕微鏡 / 化学修飾 / PEG化修飾 / 構造生物学 / アモルファス / 気液界面 / グリッド
Outline of Research at the Start

クライオ電顕は構造解析の主要な方法論となりつつあるが、電顕観察に供するグリッド作製に関しては困難を伴うことが多い。サンプル凍結の際のアグリゲーション、変性、粒子の分散性の悪化などである。グリッド上での粒子の挙動を改善するには多大な労力を要するのが一般的である。本研究課題において、PEG化手法をクライオ電子顕微鏡解析のグリッド作製に適用することで、上記の困難を画一的に克服する手法を提供する可能性がある。

Outline of Final Research Achievements

Cryo-EM analysis has become a major methodology for protein structural analysis in recent years. However, improving behaviors of protein particles on the cryo-EM grids often poses a bottleneck in structural analysis. In this study, I aimed to improve the behaviors of protein particles on cryo-EM grid by PEGylating proteins. All of the model proteins tested showed improved particle dispersion for further structural analyses. This method could be widely used as a versatile and effective option for cryo-EM grid preparation.

Academic Significance and Societal Importance of the Research Achievements

タンパク質の構造情報は、生命現象を理解しその制御方法を考えるうえで、近年ますます重要になってきている。タンパク質の構造情報を実験的に得る方法として、クライオ電子顕微鏡解析が盛んに行われているが、構造解析に適したグリッドを調製する段階がボトルネックになることが多い。本研究では、タンパク質をPEG化することでグリッド上での粒子の挙動が改善することを示した。本手法は、クライオ電子顕微鏡構造解析をさらにスピードアップさせるとともに、これまで構造解析できなかった試料についても新たな可能性を提供するものである。

Report

(3 results)
  • 2022 Annual Research Report   Final Research Report ( PDF )
  • 2021 Research-status Report
  • Research Products

    (13 results)

All 2023 2022 2021 Other

All Journal Article (10 results) (of which Int'l Joint Research: 4 results,  Peer Reviewed: 10 results,  Open Access: 7 results) Presentation (1 results) Remarks (2 results)

  • [Journal Article] Novel multivalent S100A8 inhibitory peptides attenuate tumor progression and metastasis by inhibiting the TLR4-dependent pathway2023

    • Author(s)
      Deguchi A, Watanabe-Takahashi M, Mishima T, Omori T, Ohto U, Arashiki N, Nakamura F, Nishikawa K, Maru Y.
    • Journal Title

      Cancer Gene Ther

      Volume: - Issue: 7 Pages: 1-12

    • DOI

      10.1038/s41417-023-00604-3

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction2023

    • Author(s)
      Sakaniwa Kentaro、Fujimura Akiko、Shibata Takuma、Shigematsu Hideki、Ekimoto Toru、Yamamoto Masaki、Ikeguchi Mitsunori、Miyake Kensuke、Ohto Umeharu、Shimizu Toshiyuki
    • Journal Title

      Nature Communications

      Volume: 14 Issue: 1 Pages: 164-164

    • DOI

      10.1038/s41467-023-35844-2

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Activation and regulation mechanisms of NOD-like receptors based on structural biology2022

    • Author(s)
      Ohto Umeharu
    • Journal Title

      Frontiers in Immunology

      Volume: 13 Pages: 953530-953530

    • DOI

      10.3389/fimmu.2022.953530

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Structure of SARS-CoV-2 membrane protein essential for virus assembly2022

    • Author(s)
      Zhang Zhikuan、Nomura Norimichi、Muramoto Yukiko、Ekimoto Toru、Uemura Tomoko、Liu Kehong、Yui Moeko、Kono Nozomu、Aoki Junken、Ikeguchi Mitsunori、Noda Takeshi、Iwata So、Ohto Umeharu、Shimizu Toshiyuki
    • Journal Title

      Nature Communications

      Volume: 13 Issue: 1 Pages: 4399-4399

    • DOI

      10.1038/s41467-022-32019-3

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Propofol directly binds to and inhibits <scp>TLR7</scp>2022

    • Author(s)
      Koutsogiannaki Sophia、Bu Weiming、Maisat Wiriya、Manzor Mariel、Zhang Zhikuan、Ohto Umeharu、Eckenhoff Roderic G.、Yuki Koichi
    • Journal Title

      The FASEB Journal

      Volume: 36 Issue: 8

    • DOI

      10.1096/fj.202200312r

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Structure of the?bile acid transporter?and HBV receptor NTCP2022

    • Author(s)
      Asami Jinta、Kimura Kanako Terakado、Fujita-Fujiharu Yoko、Ishida Hanako、Zhang Zhikuan、Nomura Yayoi、Liu Kehong、Uemura Tomoko、Sato Yumi、Ono Masatsugu、Yamamoto Masaki、Noda Takeshi、Shigematsu Hideki、Drew David、Iwata So、Shimizu Toshiyuki、Nomura Norimichi、Ohto Umeharu
    • Journal Title

      Nature

      Volume: 606 Issue: 7916 Pages: 1021-1026

    • DOI

      10.1038/s41586-022-04845-4

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation2022

    • Author(s)
      Ohto Umeharu、Kamitsukasa Yukie、Ishida Hanako、Zhang Zhikuan、Murakami Karin、Hirama Chie、Maekawa Sakiko、Shimizu Toshiyuki
    • Journal Title

      Proceedings of the National Academy of Sciences

      Volume: 119 Issue: 11

    • DOI

      10.1073/pnas.2121353119

    • Related Report
      2021 Research-status Report
    • Peer Reviewed
  • [Journal Article] The structure of NLRP9 reveals a unique C‐terminal region with putative regulatory function2022

    • Author(s)
      Kamitsukasa Yukie、Nakano Kenji、Murakami Karin、Hirata Kunio、Yamamoto Masaki、Shimizu Toshiyuki、Ohto Umeharu
    • Journal Title

      FEBS Letters

      Volume: 596 Issue: 7 Pages: 876-885

    • DOI

      10.1002/1873-3468.14302

    • Related Report
      2021 Research-status Report
    • Peer Reviewed
  • [Journal Article] Tetrasubstituted imidazoles as incognito Toll-like receptor 8 a(nta)gonists2021

    • Author(s)
      Yang Yi、Csakai Adam、Jiang Shuangshuang、Smith Christina、Tanji Hiromi、Huang Jian、Jones Torey、Sakaniwa Kentaro、Broadwell Lindsey、Shi Chengrui、Soti Subada、Ohto Umeharu、Fang Yaohui、Shen Shu、Deng Fei、Shimizu Toshiyuki、Yin Hang
    • Journal Title

      Nature Communications

      Volume: 12 Issue: 1 Pages: 4351-4351

    • DOI

      10.1038/s41467-021-24536-4

    • Related Report
      2021 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Improving particle quality in cryo-EM analysis using a PEGylation method2021

    • Author(s)
      Zhang Zhikuan、Shigematsu Hideki、Shimizu Toshiyuki、Ohto Umeharu
    • Journal Title

      Structure

      Volume: 29 Issue: 10 Pages: 1192-1199.e4

    • DOI

      10.1016/j.str.2021.05.004

    • Related Report
      2021 Research-status Report
    • Peer Reviewed
  • [Presentation] B型肝炎ウイルスの受容体の立体構造とウイルス感染機構2023

    • Author(s)
      大戸梅治
    • Organizer
      日本薬学会第143年会
    • Related Report
      2022 Annual Research Report
  • [Remarks] 研究室HP

    • URL

      https://kouzou.f.u-tokyo.ac.jp/

    • Related Report
      2022 Annual Research Report
  • [Remarks] 研究室ホームページ

    • URL

      https://kouzou.f.u-tokyo.ac.jp/

    • Related Report
      2021 Research-status Report

URL: 

Published: 2021-07-13   Modified: 2024-01-30  

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