1. Structural Biology and Molecular Biophysics

Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states

  1. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart  Is a corresponding author
  1. The Victor Chang Cardiac Research Institute, Australia
  2. Nanyang Technological University, Singapore
  3. University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.21598
Share this article
Cite this article
  1. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart
(2016)
Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states
eLife 5:e21598.
https://doi.org/10.7554/eLife.21598
  2. Download BibTeX
  3. Download .RIS

Abstract

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the <em>E. coli</em> F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk's &epsilon; subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30&ordm; to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates towards the membrane with its helices separating to embrace subunit a from two sides.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Meghna Sobti

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  2. Callum Smits

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  3. Andrew SW Wong

    NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.

  4. Robert Ishmukhametov

    Department of Physics, Clarendon Laboratory, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Daniela Stock

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    Competing interests
    The authors declare that no competing interests exist.

  6. Sara Sandin

    NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore
    Competing interests
    The authors declare that no competing interests exist.

  7. Alastair G Stewart

    Molecular, Structural and Computational Biology Division, The Victor Chang Cardiac Research Institute, Darlinghurst, Australia
    For correspondence
    a.stewart@victorchang.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2070-6030

Funding

National Health and Medical Research Council (1004620)

  • Daniela Stock

National Health and Medical Research Council (1109961)

  • Daniela Stock

National Health and Medical Research Council (1090408)

  • Alastair G Stewart

National Health and Medical Research Council (1022143)

  • Daniela Stock

National Health and Medical Research Council (1047004)

  • Daniela Stock

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Werner Kühlbrandt, Max Planck Institute of Biophysics, Germany

Publication history

  1. Received: September 19, 2016
  2. Accepted: December 15, 2016
  3. Accepted Manuscript published: December 21, 2016 (version 1)
  4. Accepted Manuscript updated: December 22, 2016 (version 2)
  5. Version of Record published: January 5, 2017 (version 3)
  6. Version of Record updated: February 10, 2017 (version 4)

Copyright

© 2016, Sobti et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,701
    Page views
  • 889
    Downloads
  • 85
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Meghna Sobti
  2. Callum Smits
  3. Andrew SW Wong
  4. Robert Ishmukhametov
  5. Daniela Stock
  6. Sara Sandin
  7. Alastair G Stewart
(2016)
Cryo-EM structures of the autoinhibited <em>E. coli</em> ATP synthase in three rotational states
eLife 5:e21598.
https://doi.org/10.7554/eLife.21598

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

    Meghna Sobti et al.
    Research Advance Updated

    ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, β and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate ‘half-up’ state to a condensed ‘down’ state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory ‘up’ state.

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics

    Structure of the IL-27 quaternary receptor signaling complex

    Nathanael A Caveney et al.
    Short Report Updated

    Interleukin 27 (IL-27) is a heterodimeric cytokine that functions to constrain T cell-mediated inflammation and plays an important role in immune homeostasis. Binding of IL-27 to cell surface receptors, IL-27Rα and gp130, results in activation of receptor-associated Janus Kinases and nuclear translocation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT3 transcription factors. Despite the emerging therapeutic importance of this cytokine axis in cancer and autoimmunity, a molecular blueprint of the IL-27 receptor signaling complex, and its relation to other gp130/IL-12 family cytokines, is currently unclear. We used cryogenic-electron microscopy to determine the quaternary structure of IL-27, composed of p28 and Epstein-Barr Virus-Induced 3 (Ebi3) subunits, bound to receptors, IL-27Rα and gp130. The resulting 3.47 Å resolution structure revealed a three-site assembly mechanism nucleated by the central p28 subunit of the cytokine. The overall topology and molecular details of this binding are reminiscent of IL-6 but distinct from related heterodimeric cytokines IL-12 and IL-23. These results indicate distinct receptor assembly mechanisms used by heterodimeric cytokines with important consequences for targeted agonism and antagonism of IL-27 signaling.

    1. Structural Biology and Molecular Biophysics

    Deciphering a hexameric protein complex with Angstrom optical resolution

    Hisham Mazal et al.
    Research Article

    Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.