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Related Experiment Videos

Asymmetry and structural changes in ECF1 examined by cryoelectronmicroscopy

S Wilkens1, R A Capaldi

  • 1Institute of Molecular Biology, University of Oregon, Eugene 97403.

Biological Chemistry Hoppe-Seyler
|January 1, 1994
PubMed
Summary

Cryo-EM revealed asymmetry in Escherichia coli ATPase (ECF1). The study mapped the locations of gamma and epsilon subunits, showing nucleotide-dependent positional shifts for both, crucial for enzyme function.

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Area of Science:

  • Structural Biology
  • Biochemistry
  • Molecular Motors

Background:

  • The Escherichia coli F1-ATPase (ECF1) is a key enzyme responsible for ATP hydrolysis.
  • Understanding the spatial arrangement and dynamics of its subunits is critical for elucidating its mechanism.
  • Previous studies indicated potential movement of subunits during catalysis.

Purpose of the Study:

  • To determine the three-dimensional structure and subunit arrangement of ECF1 using cryo-electron microscopy.
  • To investigate the positional dynamics of the gamma and epsilon subunits within the ECF1 complex.
  • To explore the nucleotide dependence of subunit positioning.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) was employed to visualize the ECF1 complex.

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  • Image analysis techniques were used to distinguish individual beta subunits based on projection density.
  • Site-directed mutagenesis and gold particle labeling were utilized to track the epsilon subunit's position.

    • Main Results:

    • Intrinsic asymmetry was identified in the hexagonal projection of ECF1, allowing differentiation of the three beta subunits (β1, β2, β3).
    • The gamma subunit's position was not fixed, exhibiting an arc-like shift of approximately 10Å relative to the center of mass, potentially dependent on nucleotide binding.
    • The epsilon subunit was localized to an arc between an alpha and a beta subunit, with a shift of approximately 20Å, and its position demonstrated nucleotide dependence.

    Conclusions:

    • The study successfully mapped the positions of the gamma and epsilon subunits within the ECF1 complex.
    • Evidence is presented for nucleotide-dependent positional changes of the epsilon subunit, complementing known nucleotide dependence of the gamma subunit.
    • These findings provide insights into the conformational flexibility and dynamic mechanism of rotary ATPases.