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CryoEM and image sorting for flexible protein/DNA complexes.

Seth A Villarreal1, Phoebe L Stewart1

  • 1Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, United States.

Journal of Structural Biology
|December 17, 2013
PubMed
Summary
This summary is machine-generated.

Eigenimage sorting analysis helped visualize flexible protein-DNA complexes involved in DNA repair. This method revealed density for DNA, streptavidin, and Ku near the base of DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

Keywords:
Cryo-electron microscopyCryoEMEigenimage sortingFlexibilityHeterogeneityProtein/DNA complexesSingle particle reconstruction

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Area of Science:

  • Structural biology
  • Biochemistry
  • Molecular genetics

Background:

  • Protein complexes with intrinsically disordered regions and conformational flexibility are crucial for biological functions.
  • Disorder and flexibility in molecular complexes often pose challenges for structural analysis.
  • Cryo-electron microscopy (CryoEM) and single particle image processing enable imaging of flexible samples.

Purpose of the Study:

  • To demonstrate the utility of eigenimage sorting for analyzing protein-DNA complexes with significant flexibility and heterogeneity.
  • To investigate the structural organization of DNA-PKcs complexes involved in DNA double-strand break repair.

Main Methods:

  • Cryo-electron microscopy (CryoEM) and single particle image processing.
  • Eigenimage sorting analysis to divide heterogeneous particle images into more homogeneous subsets.
  • 3D reconstruction and comparison of subset structures with 2D class averages.

Main Results:

  • Initial 3D reconstructions of DNA-PKcs complexes lacked clear density for bound components.
  • Eigenimage sorting facilitated the division of datasets into homogeneous subsets.
  • Visualization of additional density near the DNA-PKcs base, attributed to DNA, streptavidin, and Ku.

Conclusions:

  • Eigenimage sorting is a valuable tool for analyzing flexible and heterogeneous protein complexes.
  • DNA appears to bind near the base of DNA-PKcs, potentially interacting with a flexible region.
  • Further refinement is needed to resolve the complete structure due to remaining heterogeneity.