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

Abstract
Data availability
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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. Braig K
2. Otwinowski Z
3. Hegde R
4. Boisvert DC
5. Joachimiak A
6. Horwich AL
7. Sigler
8. PB
(1995) THE CRYSTAL STRUCTURE OF THE BACTERIAL CHAPERONIN GROEL AT 2.8 ANGSTROMS
Publicly available at the RCSB Protein Data Bank (accession no: 1GRL).

http://www.rcsb.org/pdb/explore/explore.do?structureId=1GRL
1. Estrozi LF
2. Settembre EC
3. Goret G
4. McClain B
5. Zhang X
6. Chen JZ
7. Grigorieff N
8. Harrison SC
(2012) Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles
Publicly available at the RCSB Protein Data Bank (accession no: 4F5X).

http://www.rcsb.org/pdb/explore/explore.do?structureId=4F5X
1. Lu X
2. Harrison SC
3. Tao YJ
4. Patton JT
5. Nibert ML
(2008) Crystal Structure of VP1 apoenzyme of Rotavirus SA11 (N-terminal hexahistidine-tagged)
Publicly available at the RCSB Protein Data Bank (accession no: 2R7O).

http://www.rcsb.org/pdb/explore/explore.do?structureId=2R7O
(2009) Crystal Structure of the Rotavirus Double Layered Particle
Publicly available at the RCSB Protein Data Bank (accession no: 3KZ4).

http://www.rcsb.org/pdb/explore/explore.do?structureId=3KZ4
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

J Peter Rickgauer

Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States

Competing interests
The authors declare that no competing interests exist.
Nikolaus Grigorieff

Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1506-909X
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.

"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

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.