Challenge to near atomic resolution AFM image by fusion of high-speed AFM and advanced image analysis

• Project/Area Number: 18K19287
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Kanazawa University
• Principal Investigator: Mikihiro Shibata 金沢大学, ナノ生命科学研究所, 准教授 (80631027)
• Project Period (FY): 2018-06-29 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2019: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000); Fiscal Year 2018: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
• Keywords: タンパク質 / バイオイメージング / 原子間力顕微鏡 / 画像解析

Outline of Final Research Achievements

High-speed atomic force microscopy (HS-AFM) is a unique technique to capture a nano-dynamics of biomolecules under the near physiological conditions. The purpose of this study is to build the image analysis which is capable to quantify fluctuation and structural changes of proteins by combining with single-particle analysis and deep learning. Specifically, we applied MotionCor2 to HS-AFM movies of proteins. As a result, we succeeded in drift-correcting about 200 AFM images with a sub-nanometer accuracy and obtained the integrated image. In the integrated image, the spatial resolution is improved in the part where the protein has a little fluctuation, while the resolution is decreased in the part where the fluctuation is large. Thus, this new combined method allows us to quantify a fluctuation of proteins.

Academic Significance and Societal Importance of the Research Achievements

ほとんど全ての生命現象はタンパク質の作用に基づいており、それ故、タンパク質がはたらく仕組みを理解することは、生命科学において非常に重要である。近年の生命科学において、タンパク質の機能発現には、タンパク質部位の“動き”や“揺らぎ”が重要であることが報告され、真にタンパク質の仕組みを理解するには、動きまで含めた動的な構造解析が必須である。本研究は、タンパク質のナノ動態を直接可視化できる高速AFM画像に対し、様々な画像解析を適用するシステムを構築したことにより、高速AFM画像において、これまで見過ごしていたタンパク質の”揺らぎ“に関する情報をより客観的な解析により引き出せるようになった。

Research Products

• [Journal Article] Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor. (2019)
  • Author(s): Sakai K, Passioura T, Sato H, Ito K, Furuhashi H, Umitsu M, Takagi J, Kato Y, Mukai H, Warashina S, Zouda M, Watanabe Y, Yano S, Shibata M, Suga H, Matsumoto K
  • Journal Title: Nature Chemical Biology Volume: 15 Issue: 6 Pages: 598-606
  • DOI: 10.1038/s41589-019-0285-7
  • Peer Reviewed / Open Access
• [Journal Article] Crystal structure of heliorhodopsin (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, T. Izume, Okazaki S, Hashimoto M, Mizutori R, Tomida S, Yamauchi Y, Abe-Yoshizumi R, K. Katayama, SP. Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Béjà, T. Uchihashi, H. Kandori, O. Nureki
  • Journal Title: Nature Volume: 574 Issue: 7776 Pages: 132-136
  • DOI: 10.1038/s41586-019-1604-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor. (2019)
  • Author(s): Katsuya Sakai, Toby Passioura, Hiroki Sato, Kenichiro Ito, Hiroki Furuhashi, Masataka Umitsu, Junichi Takagi, Yukinari Kato, Hidefumi Mukai, Shota Warashina, Maki Zouda, Yasuyoshi Watanabe, Seiji Yano, Mikihiro Shibata, Hiroaki Suga, and Kunio Matsumoto
  • Journal Title: Nat. Chem. Biol. Volume: 印刷中
  • Peer Reviewed
• [Journal Article] Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy (2018)
  • Author(s): Shibata Mikihiro、Inoue Keiichi、Ikeda Kento、Konno Masae、Singh Manish、Kataoka Chihiro、Abe-Yoshizumi Rei、Kandori Hideki、Uchihashi Takayuki
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 8262-8262
  • DOI: 10.1038/s41598-018-26606-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] High thermal stability of oligomeric assemblies of Thermophilic rhodopsin in a lipid environment. (2018)
  • Author(s): Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, and Yasuhisa Mizutani
  • Journal Title: J. Phys. Chem. B Volume: 122 Issue: 27 Pages: 6945-6953
  • DOI: 10.1021/acs.jpcb.8b04894
  • Peer Reviewed
• [Presentation] Signal integration mechanism of Ca2+/calmodulin-dependent protein kinase II revealed by High-speed AFM. (2020)
  • Author(s): Mikihiro Shibata and Hideji Murakoshi
  • Organizer: BPS2020 64th Annual Meeting
  • Int'l Joint Research
• [Presentation] Nano-scale imaging of biological samples using High-speed Atomic Force Microscopy. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: LIIF2019
  • Int'l Joint Research / Invited
• [Presentation] 高速原子間力顕微鏡によるクロマチン動態のナノスケール観察 (2019)
  • Author(s): 柴田幹大
  • Organizer: 第２回クロマチン潜在能領域会議
  • Invited
• [Presentation] Visualizing flexibility in protein structures by high-speed atomic force microscopy. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: 57th日本生物物理学会年会
  • Invited
• [Presentation] High-speed atomic force microscopy visualization of protein flexibility in action. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: Joint UBI-NanoLSI workshop TRENDS IN MOLECULAR BIOPHYSICS OF LIVING CELLS
  • Invited
• [Presentation] Physicochemical properties of the microbial rhodopsin RxR in the membrane-mimicking molecule styrene maleic acid (SMA) copolymer. (2019)
  • Author(s): Tetsuya Ueta, Keiichi Kojima, Tomoya Hino, Mikihiro Shibata, Shingo Nagano & Yuki Sudo
  • Organizer: 第11回日本生物物理学会 中国四国支部大会
• [Presentation] Structure and biophysical characterization of the heliorhodopsin. (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, S. Okazaki, T. Izume, M. Hashimoto, R. Mizutori, S. Tomida, Y. Yamauchi, R. Abe-Yoshizumi, K. Katayama, S. P.Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Beja, T. Uchihashi, H. Kandori and O. Nureki
  • Organizer: 57th日本生物物理学会年会
• [Presentation] Application of drift elimination method for high-speed AFM images. (2019)
  • Author(s): Shotaro Tsujioka, Hideji Murakoshi, and Mikihiro Shibata
  • Organizer: 57th日本生物物理学会年会
• [Presentation] Mapping of mechanical property on live cell surface by scanning ion conductance microscope. (2019)
  • Author(s): Satoko Kitazawa, Linhao Sun, Ayako Housaka, Takahiro Watanabe-Nakayama, Hiroki Konno, Mikihiro Shibata, and Shinji Watanabe
  • Organizer: 57th日本生物物理学会年会
• [Presentation] High-speed atomic force microscopy shows conformational dynamics of Ca2+/calmodulin-dependent protein kinase II (2019)
  • Author(s): Mikihiro Shibata and Hideji Murakoshi
  • Organizer: BPS19 63th Annual Meeting
• [Presentation] 高速原子間力顕微鏡による生体分子のナノスケールイメージング (2018)
  • Author(s): 柴田幹大
  • Organizer: 2018日本表面真空学会学術講演会
  • Invited
• [Presentation] 高速原子間力顕微鏡による揺らいだタンパク質構造のナノスケール撮影 (2018)
  • Author(s): 柴田幹大
  • Organizer: 非共有結合系の分子科学：計測技術から探る生体分子科学の新展開
  • Invited
• [Presentation] 計測系から見た分子集合体の機能計測-高速AFM計測 (2018)
  • Author(s): 柴田幹大
  • Organizer: 日本化学会第99秋季年会 複雑系のための分子科学―集まって立ち現れる分子機能の理解と設計
  • Invited