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Single-protein detection in crowded molecular environments in cryo-EM images
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 spe...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453696/ https://www.ncbi.nlm.nih.gov/pubmed/28467302 http://dx.doi.org/10.7554/eLife.25648 |
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author | Rickgauer, J Peter Grigorieff, Nikolaus Denk, Winfried |
author_facet | Rickgauer, J Peter Grigorieff, Nikolaus Denk, Winfried |
author_sort | Rickgauer, J Peter |
collection | PubMed |
description | 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. DOI: http://dx.doi.org/10.7554/eLife.25648.001 |
format | Online Article Text |
id | pubmed-5453696 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-54536962017-06-02 Single-protein detection in crowded molecular environments in cryo-EM images Rickgauer, J Peter Grigorieff, Nikolaus Denk, Winfried eLife Biophysics and Structural Biology 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. DOI: http://dx.doi.org/10.7554/eLife.25648.001 eLife Sciences Publications, Ltd 2017-05-03 /pmc/articles/PMC5453696/ /pubmed/28467302 http://dx.doi.org/10.7554/eLife.25648 Text en © 2017, Rickgauer et al http://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biophysics and Structural Biology Rickgauer, J Peter Grigorieff, Nikolaus Denk, Winfried Single-protein detection in crowded molecular environments in cryo-EM images |
title | Single-protein detection in crowded molecular environments in cryo-EM images |
title_full | Single-protein detection in crowded molecular environments in cryo-EM images |
title_fullStr | Single-protein detection in crowded molecular environments in cryo-EM images |
title_full_unstemmed | Single-protein detection in crowded molecular environments in cryo-EM images |
title_short | Single-protein detection in crowded molecular environments in cryo-EM images |
title_sort | single-protein detection in crowded molecular environments in cryo-em images |
topic | Biophysics and Structural Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453696/ https://www.ncbi.nlm.nih.gov/pubmed/28467302 http://dx.doi.org/10.7554/eLife.25648 |
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