Journal: J Struct Biol / Year: 2014 Title: CryoEM and image sorting for flexible protein/DNA complexes. Authors: Seth A Villarreal / Phoebe L Stewart / Abstract: Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder ...Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.
History
Deposition
Dec 10, 2013
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Header (metadata) release
Jan 1, 2014
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Map release
Jan 1, 2014
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Update
Jul 16, 2014
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Current status
Jul 16, 2014
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: Tecnai Polara / Image courtesy: FEI Company
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Image processing
Details
The particles were selected with an in-house script and processed using Frealign and 3D Fourier Space v2.
CTF correction
Details: Each micrograph
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.4 Å / Resolution method: OTHER / Software - Name: Frealign, Imagic / Number images used: 19825
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