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

Structure of a bacterial ATP synthase

  1. Hui Guo
  2. Toshiharu Suzuki
  3. John L Rubinstein  Is a corresponding author
  1. The Hospital for Sick Children, Canada
  2. Tokyo Institute of Technology, Japan
Research Article
  • Cited 56
  • Views 19,548
  • Annotations
Cite this article as: eLife 2019;8:e43128 doi: 10.7554/eLife.43128

Abstract

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit e shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

Data availability

CryoEM maps have been deposited in EMDB and atomic models in PDB.

The following data sets were generated
    1. Guo H
    2. Rubinstein JL
    (2018) Intact class 1
    Electron Microscopy Data Bank, EMD-9333.
    1. Guo H
    2. Rubinstein JL
    (2018) Intact class 2
    Electron Microscopy Data Bank, EMD-9334.
    1. Guo H
    2. Rubinstein JL
    (2018) Intact class 3
    Electron Microscopy Data Bank, EMD-9335.
    1. Guo H
    2. Rubinstein JL
    (2018) Focused Fo
    Electron Microscopy Data Bank, EMD-9327.

Article and author information

Author details

  1. Hui Guo

    Molecular Medicine, The Hospital for Sick Children, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. Toshiharu Suzuki

    Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.
  3. John L Rubinstein

    Molecular Medicine, The Hospital for Sick Children, Toronto, Canada
    For correspondence
    john.rubinstein@utoronto.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0566-2209

Funding

Canadian Institutes of Health Research (MOP 81294)

  • John L Rubinstein

Canada Research Chairs

  • John L Rubinstein

Japan Society for the Promotion of Science (JP18H02409)

  • Toshiharu Suzuki

Canada Foundation for Innovation

  • John L Rubinstein

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

Reviewing Editor

  1. Richard M Berry, University of Oxford, United Kingdom

Publication history

  1. Received: October 26, 2018
  2. Accepted: February 2, 2019
  3. Accepted Manuscript published: February 6, 2019 (version 1)
  4. Version of Record published: February 15, 2019 (version 2)

Copyright

© 2019, Guo 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

  • 19,548
    Page views
  • 1,377
    Downloads
  • 56
    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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    Dhruva Katrekar et al.
    Tools and Resources

    Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their enzymatic activity on adenosines flanked by a 5' guanosine is very low, thus limiting their utility as a transcriptome engineering toolset. To address this issue, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. However, exogenous delivery of ADAR enzymes, especially hyperactive variants, leads to significant transcriptome wide off-targeting. To solve this problem, we engineered a split ADAR2 deaminase which resulted in 1000-fold more specific RNA editing as compared to full-length deaminase overexpression. We anticipate that this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Keith F DeLuca et al.
    Tools and Resources Updated

    Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.