Investigation into Fo/F1 coupling of the FoF1 ATP synthase by time resolved single particle cryo-EM

• Project/Area Number: 15K14464
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: University of Hyogo
• Principal Investigator: Gerle Christoph 兵庫県立大学, 生命理学研究科, 特任准教授 (10561970)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: membrane protein / molecular machine / rotational catalysis / mitochondria / single particle cryo-EM / structural dynamics / sample preparation / protein purification / cryo-EM / single particle / image processing

Outline of Final Research Achievements

Mitochondrial FoF1-ATP synthase is central to energy conversion in all mammalian cells and employs a highly efficient mechanism of rotary catalysis to recycle cellular ATP from ADP and Pi. The structural basis for the efficient coupling between membrane bound Fo domain and matrix F1 domain remains unresolved due to the absence of X-ray crystal structures of the intact enzyme and the difficulty of visualizing large scale movements by X-ray crystallography in general. Here we took up the challenge of using the emerging technique of single particle cryo-EM to tacke this important biological problem. Purification of the bovine enzyme from cow heart muscle tissue was successfully established yielding more than 100 mg of excellent sample on a reproducible basis. A novel sample preparation approach named GraDeR was developed and successfully applied to monomeric bovine FoF1 ATP synthase and first structures obtained at a resolution of ~11 Angstrom.

Research Products

• [Journal Article] Recent advances of single particle analysis by cryo-electron microscopy and its application to membrane proteins (2016)
  • Author(s): 光岡薫, Gerle, Christoph
  • Journal Title: 生化学 Volume: 88 Issue: 4 Pages: 532-536
  • DOI: 10.14952/SEIKAGAKU.2016.880532
  • NAID: 40020948610
  • ISSN: 0037-1017
  • Year and Date: 2016-08-25
  • Peer Reviewed
• [Journal Article] Osuda Y, Shinzawa-Itoh K, Tani K, Maeda S, Yoshikawa S, Tsukihara T, Gerle C. (2016)
  • Author(s): Osuda Y, Shinzawa-Itoh K, Tani K, Maeda S, Yoshikawa S, Tsukihara T, Gerle C.
  • Journal Title: Microscopy Volume: 65 Issue: 3 Pages: 263-267
  • DOI: 10.1093/jmicro/dfv381
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] On the structural possibility of pore-forming mitochondrial FoF1 ATP synthase (2016)
  • Author(s): Gerle, Christoph
  • Journal Title: BBA - Bioenergetics Volume: in press Issue: 8 Pages: 1191-1196
  • DOI: 10.1016/j.bbabio.2016.03.008
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] GraDeR: membrane protein complex preparation for single particle cryo-EM. (2015)
  • Author(s): Hauer F., Gerle C., Fischer N., Oshima A., Shinzawa-Itoh K., Shimada S., Yokoyama K., Fujiyoshi Y. & Stark H.
  • Journal Title: Structure Volume: 23 Issue: 9 Pages: 1769-1775
  • DOI: 10.1016/j.str.2015.06.029
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Bovine F1FoATP synthase monomers bend the lipid bilayer in 2D membrane crystals. (2015)
  • Author(s): C. Jiko, K.M. Davies, K.S. Itoh, K. Tani, S. Maeda, D.J. Mills, T. Tsukihara, Y. Fujiyoshi W. Kuehlbrandt and C. Gerle
  • Journal Title: eLIFE Volume: 06119 Pages: 1-36
  • DOI: 10.7554/elife.06119
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] GraDeR:単粒子解析等の膜タンハク質資料調整 (2016)
  • Author(s): Christoph Gerle, Florian Hauer, Niels Fischer, Kyoko Shinzawa-Itoh, Satoru Shimada, Ken Yokoyama, Atsunori Oshima, Yoshinori Fujiyoshi, Holger Stark
  • Organizer: 日本生物物理学会
  • Place of Presentation: つくば国際会議場（茨城県、つくば市）
  • Year and Date: 2016-11-25
• [Presentation] On the structural possibility of pore forming mitochondrial FoF1 ATP synthase. (2016)
  • Author(s): Gerle, C.
  • Organizer: EBEC
  • Place of Presentation: Riva del Garda, Italy
  • Year and Date: 2016-07-02
  • Int'l Joint Research / Invited
• [Presentation] Electron crystallography of tomographic volumes. (2016)
  • Author(s): Kazutoshi Tani, Karen Davies, Chimari Jiko, Shintaro Maeda, Kyoko Shinzawa-Itoh, Deryck Mills, Tomitake Tsukihara, Yoshinori Fujiyoshi, Werner Kuhlbrandt, Christoph Gerle
  • Organizer: 3DEM Gordon Research Conference
  • Place of Presentation: Hong Kong University, Hong Kong
  • Year and Date: 2016-06-19
  • Int'l Joint Research
• [Presentation] Looking at a cellular machine in & out of the membrane: cryo-EM of the mammalian FoF1 ATP synthase. (2016)
  • Author(s): Christoph Gerle
  • Organizer: IPR Seminar 'Introduction and overview of cryo-electron microscopy'
  • Place of Presentation: 大阪大学 (大阪府吹田市)
  • Year and Date: 2016-02-19
  • Invited
• [Presentation] In & out of the membrane: cryoEM of bovine FoF1 ATP synthase. (2016)
  • Author(s): Gerle, C.
  • Organizer: Minisymposium on Structure-Based Bioenergetics
  • Place of Presentation: Spring-8 (兵庫県、佐用郡）
  • Invited
• [Presentation] Membrane bending and more: cryoEM of the mammalian F-ATP synthase (2016)
  • Author(s): Gerle, C.
  • Organizer: Mitochondrial bASiCs: ATP, Signaling and Calcium symposium
  • Place of Presentation: University of Padua, Italy
  • Int'l Joint Research / Invited
• [Presentation] CryoEM of the mammalian F-ATP synthase: possible implications for the mitochondrial permeability transition pore. (2016)
  • Author(s): Gerle, C.
  • Organizer: departmental seminar
  • Place of Presentation: University of Padua, Italy
  • Invited
• [Presentation] Novel sample preparation for cryo-EM: in silico & in aqua. (2015)
  • Author(s): Christoph Gerle
  • Organizer: Young Scientists Satellite Meeting - Toward Next Generation of Microscopic Sciences - in The 2nd East-Asia Microscopy Conference
  • Place of Presentation: 淡路夢舞台（兵庫県淡路市）
  • Year and Date: 2015-11-27
  • Int'l Joint Research / Invited
• [Presentation] In & Out of the Membrane: EM structure of the mammalian FoF1 ATP synthase (2015)
  • Author(s): Christoph Gerle
  • Organizer: Cold Spring Harbor Asia conference on Mitochondria
  • Place of Presentation: Suzhou, China
  • Year and Date: 2015-10-12
  • Int'l Joint Research / Invited
• [Presentation] Looking at "bad" 2D crystals can be good. (2015)
  • Author(s): Christoph Gerle
  • Organizer: Seminar Talk, LMB, Cambridge, U.K.
  • Place of Presentation: Cambridge, U.K.
  • Year and Date: 2015-07-03
  • Int'l Joint Research / Invited
• [Presentation] GraDeR: membrane protein complex preparation for single particle cryo-EM. (2015)
  • Author(s): Gerle C., Hauer F., Fischer N., Oshima A., Shinzawa-Itoh K., Shimada S., Yokoyama K., Fujiyoshi Y. & Stark H.
  • Organizer: 3DEM Gordon Research Conference
  • Place of Presentation: Colby-Sawyer College, New London. NH, U.S.A.
  • Year and Date: 2015-06-21
  • Int'l Joint Research
• [Presentation] GraDeR: membrane protein complex preparation for single particle cryo-EM. (2015)
  • Author(s): Christoph Gerle
  • Organizer: 71st annual meeting of the Japanese Society of Microscopy
  • Place of Presentation: 京都国際会館（京都府京都市）
  • Year and Date: 2015-05-13
  • Invited
• [Remarks] GraDeR for micelle-free membrane proteins
  • URL: https://grader-protocol.com/