Single-protein detection in crowded molecular environments in cryo-EM images
- Howard Hughes Medical Institute, Janelia Research Campus, United States
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
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Horse spleen apoferritinPublicly available at the RCSB Protein Data Bank (accession no: 2W0O).
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THE CRYSTAL STRUCTURE OF THE BACTERIAL CHAPERONIN GROEL AT 2.8 ANGSTROMSPublicly available at the RCSB Protein Data Bank (accession no: 1GRL).
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Location of the dsRNA-dependent polymerase, VP1, in rotavirus particlesPublicly available at the RCSB Protein Data Bank (accession no: 4F5X).
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Crystal Structure of VP1 apoenzyme of Rotavirus SA11 (N-terminal hexahistidine-tagged)Publicly available at the RCSB Protein Data Bank (accession no: 2R7O).
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Crystal Structure of the Rotavirus Double Layered ParticlePublicly available at the RCSB Protein Data Bank (accession no: 3KZ4).
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Crystal Structure of Bovine Serum AlbuminPublicly available at the RCSB Protein Data Bank (accession no: 4F5S).
Article and author information
Author details
Nikolaus Grigorieff
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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Single-protein detection in crowded molecular environments in cryo-EM images
eLife 6:e25648.
https://doi.org/10.7554/eLife.25648
Further reading
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