1. Structural Biology and Molecular Biophysics
  2. Cell Biology

Single-protein detection in crowded molecular environments in cryo-EM images

  1. J Peter Rickgauer
  2. Nikolaus Grigorieff
  3. Winfried Denk  Is a corresponding author
  1. Howard Hughes Medical Institute, Janelia Research Campus, United States
https://doi.org/10.7554/eLife.25648
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  1. J Peter Rickgauer
  2. Nikolaus Grigorieff
  3. Winfried Denk
(2017)
Single-protein detection in crowded molecular environments in cryo-EM images
eLife 6:e25648.
https://doi.org/10.7554/eLife.25648
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Abstract

We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and-in the presence of protein background-a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.

Data availability

The following previously published data sets were used
    1. De Val N
    2. Declercq JP
    (2008) Horse spleen apoferritin
    Publicly available at the RCSB Protein Data Bank (accession no: 2W0O).
    http://www.rcsb.org/pdb/explore/explore.do?structureId=2W0O
    1. Bujacz A
    2. Bujacz G
    (2012) Crystal Structure of Bovine Serum Albumin
    Publicly available at the RCSB Protein Data Bank (accession no: 4F5S).
    http://www.rcsb.org/pdb/explore/explore.do?structureId=4F5S

Article and author information

Author details

  1. J Peter Rickgauer

    Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Nikolaus Grigorieff

    Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X

  3. Winfried Denk

    Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States
    For correspondence
    winfried.denk@neuro.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0704-6998

Funding

Howard Hughes Medical Institute (Internal)

  • J Peter Rickgauer
  • Nikolaus Grigorieff
  • Winfried Denk

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

Copyright

© 2017, Rickgauer 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.

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  1. J Peter Rickgauer
  2. Nikolaus Grigorieff
  3. Winfried Denk
(2017)
Single-protein detection in crowded molecular environments in cryo-EM images
eLife 6:e25648.
https://doi.org/10.7554/eLife.25648

Share this article

https://doi.org/10.7554/eLife.25648

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