Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus
Atsuko Nakanishi, Jun-ichi Kishikawa, Masatada Tamakoshi, Kaoru Mitsuoka & Ken Yokoyama
Nature Communications volume 9, Article number: 89 (2018)
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Received: 10 August 2017 Accepted: 04 December 2017
Published online: 08 January 2018
Abstract
Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.
Acknowledgements
We thank Dr Bernadette Byrne for critical reading of our manuscript, and members of Yokoyama Lab for help and technical assistance. This work was supported by Grants-in-Aid from “Nanotechnology Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to K.M. (Project No. 12024046) and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to K.Y. (Project No. 17H03648), and J.K. (Project No. 16K21472).
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Author notes
Atsuko Nakanishi and Jun-ichi Kishikawa contributed equally to this work.
Affiliations
Department of Molecular Biosciences, Kyoto Sangyo University, Motoyama Kamigamo, Kita-ku, Kyoto, 603-8555, Japan
Atsuko Nakanishi, Jun-ichi Kishikawa & Ken Yokoyama
Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo, 192-0392, Japan
Masatada Tamakoshi
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, 7-1, Mihogaoka, Ibaraki, Osaka, 567-0047, Japan
Kaoru Mitsuoka
Contributions
A.N., J.K. designed, performed, and analyzed the experiments. A.N., J.K., K.M. analyzed the data and contributed to the preparation of the figures. M.T. constructed vectors for expression of mutant proteins. K.M. gave technical support and conceptual advice. K.Y. designed and supervised the experiments and wrote the manuscript. All authors discussed the results and commented on the manuscript.
Competing interests
The authors declare no competing financial interests.
Corresponding authors
Correspondence to Kaoru Mitsuoka or Ken Yokoyama.
