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UVC inactivation of pathogenic samples suitable for cryoEM analysis

Depelteau, J. S. and Renault, L. and Althof, N. and Cassidy, C. K. and Mendonça, L. M. and Jensen, G. J. and Resch, G. P. and Briegel, A. (2021) UVC inactivation of pathogenic samples suitable for cryoEM analysis. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210715-140139799

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Abstract

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.07.06.451241DOIDiscussion Paper
ORCID:
AuthorORCID
Jensen, G. J.0000-0003-1556-4864
Briegel, A.0000-0003-3733-3725
Additional Information: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-NC-ND 4.0 International license. This version posted July 6, 2021. We are grateful to Davi Ortega for helpful discussions. We also wish to thank Dr. Weng Yang and Dr. Willem Noteborn of NeCEN for assistance during data collection, and Gert Koning (Department of Fine Mechanical) for assistant with the construction of the prototype UVC inactivation box. This work was funded by the Building Blocks of Life Grant 737.016.004 from the Netherlands Organization for Scientific Research and Instruct-ULTRA (Horizon 2020 Coordination and Support action Number ID: 731005.
Funders:
Funding AgencyGrant Number
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)737.016.004
European Research Council (ERC)731005
Record Number:CaltechAUTHORS:20210715-140139799
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210715-140139799
Official Citation:UVC inactivation of pathogenic samples suitable for cryoEM analysis. JS Depelteau, L Renault, N Althof, CK Cassidy, LM Mendonça, GJ Jensen, GP Resch, A Briegel. bioRxiv 2021.07.06.451241; doi: https://doi.org/10.1101/2021.07.06.451241
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109830
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Jul 2021 17:17
Last Modified:15 Jul 2021 17:17

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