Single-protein detection in crowded molecular environments in cryo-EM images
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
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.
Reviewing Editor
- Edward H Egelman, University of Virginia, United States
Publication history
- Received: February 2, 2017
- Accepted: May 2, 2017
- Accepted Manuscript published: May 3, 2017 (version 1)
- Version of Record published: June 1, 2017 (version 2)
- Version of Record updated: August 18, 2017 (version 3)
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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Further reading
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Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258–263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.
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- Cell Biology
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Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by α-actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.