Using the Volta phase plate with defocus for cryo-EM single particle analysis
- Max Planck Institute of Biochemistry, Germany
- Max Planck Institute for Biophysical Chemistry, Germany
Abstract
Previously, we reported an in-focus data acquisition method for cryo-EM single particle analysis with the Volta phase plate (VPP) (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.
Data availability
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Volta phase plate with defocus cryo-EM dataset of Thermoplasma acidophilum 20S proteasomePublicly available at the Electron Microscopy Pilot Image Archive (accession no: EMPIAR-10078).
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Thermoplasma acidophilum 20S proteasome map reconstructed with Relion 2.0 particle polishing from defocused Volta phase plate cryo-EM dataPublicly available at the EMDB Protein Data Bank in Europe (accession no: EMD-3455).
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Thermoplasma acidophilum 20S proteasome map reconstructed with MotionCor2 and Relion 2.0 from defocused Volta phase plate cryo-EM dataPublicly available at the EMDB Protein Data Bank in Europe (accession no: EMD-3456).
Article and author information
Author details
Funding
Max-Planck-Gesellschaft
- Radostin Danev
- Dimitry Tegunov
- Wolfgang Baumeister
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Sjors HW Scheres, MRC Laboratory of Molecular Biology,, United Kingdom
Version history
- Received: November 7, 2016
- Accepted: January 20, 2017
- Accepted Manuscript published: January 21, 2017 (version 1)
- Version of Record published: January 30, 2017 (version 2)
Copyright
© 2017, Danev 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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Using the Volta phase plate with defocus for cryo-EM single particle analysis
eLife 6:e23006.
https://doi.org/10.7554/eLife.23006
Further reading
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- Structural Biology and Molecular Biophysics
The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.
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- Biochemistry and Chemical Biology
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