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

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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.

DOI: http://dx.doi.org/10.7554/eLife.25648.001

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Most cited references 38

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R. Henderson (1995)

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

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R.John Ellis (2001)

Biological macromolecules have evolved over billions of years to function inside cells, so it is not surprising that researchers studying the properties of such molecules, either in extracts or in purified form, take care to control factors that reflect the intracellular environment, such as pH, ionic strength and composition, redox potential and the concentrations of relevant metabolites and effector molecules. There is one universal aspect of the cellular interior, however, that is largely neglected--the fact that it is highly crowded with macromolecules. It is proposed that the addition of crowding agents should become as routine as controlling pH and ionic strength if we are to meet the objective of studying biological molecules under more physiologically relevant conditions.

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Distributing many points on a sphere

E B Saff, A. B. J. Kuijlaars (1997)

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Author and article information

Contributors

Edward H Egelman: Role: Reviewing editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (hwp): eLife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic, pub): 03 May 2017

Publication date Collection: 2017

Volume: 6

Electronic Location Identifier: e25648

Affiliations

[1 ]Howard Hughes Medical Institute , Ashburn, United States

[2 ]deptDepartment of Electrons - Photons - Neurons , Max Planck Institute of Neurobiology , Martinsried, Germany

University of Virginia , United States

Author notes

winfried.denk@ 123456neuro.mpg.de

Author information

J Peter Rickgauer http://orcid.org/0000-0002-6957-4681

Nikolaus Grigorieff http://orcid.org/0000-0002-1506-909X

Winfried Denk http://orcid.org/0000-0002-0704-6998

Article

Publisher ID: 25648

DOI: 10.7554/eLife.25648

PMC ID: 5453696

PubMed ID: 28467302

SO-VID: 01ac45ff-ad4d-4ae4-b29e-6b12b983140c

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

History

Date received : 02 February 2017

Date accepted : 02 May 2017

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;

Award ID: Internal

Award Recipient : 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.

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elife-xml-version 2.5

Author impact statement High-resolution template matching makes small, densely embedded proteins detectable.

ScienceOpen disciplines: Life sciences

Keywords: optical methods,cryo-em,none

Data availability:

ScienceOpen disciplines: Life sciences

Keywords: optical methods, cryo-em, none

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

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Most cited references 38

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

Macromolecular crowding: an important but neglected aspect of the intracellular environment.

Distributing many points on a sphere

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Contributors

Journal

Affiliations

Author notes

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Cited by 29

Most referenced authors 679