Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Rickgauer, J Peter, Grigorieff, Nikolaus, Denk, Winfried
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2017
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
_version_ 1783240713747562496
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
work_keys_str_mv AT rickgauerjpeter singleproteindetectionincrowdedmolecularenvironmentsincryoemimages
AT grigorieffnikolaus singleproteindetectionincrowdedmolecularenvironmentsincryoemimages
AT denkwinfried singleproteindetectionincrowdedmolecularenvironmentsincryoemimages