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Fluorescence-Based Detection of Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy

View ORCID ProfileLauren Ann Metskas, View ORCID ProfileJohn A. G. Briggs
doi: https://doi.org/10.1101/388579
Lauren Ann Metskas
1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
2Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
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John A. G. Briggs
1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
2Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
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Abstract

Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperature have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD and its autoquenching by resonant energy transfer can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by resonant energy transfer can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted August 10, 2018.
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Fluorescence-Based Detection of Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy
Lauren Ann Metskas, John A. G. Briggs
bioRxiv 388579; doi: https://doi.org/10.1101/388579
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Fluorescence-Based Detection of Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy
Lauren Ann Metskas, John A. G. Briggs
bioRxiv 388579; doi: https://doi.org/10.1101/388579

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