UVC inactivation of pathogenic samples suitable for cryoEM analysis

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

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.

Authors 8

1. Jamie S. Depelteau Aachen Department of Microbial Sciences Institute of Biology, Leiden University

   Leiden University

   Affiliation as printed

   Department of Microbial Sciences, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

2. Ludovic L. R. Renault Aachen Netherlands Center for Nanoscopy (NeCEN)

   Leiden University

   Affiliation as printed

   Netherlands Center for Nanoscopy (NeCEN), Leiden University, The Netherlands

3. Nynke Althof Aachen Department of Microbial Sciences Institute of Biology, Leiden University

   Leiden University

   Affiliation as printed

   Department of Microbial Sciences, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

4. CK Cassidy

   University of Oxford

   Affiliation as printed

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

5. LM Mendonça

   California Institute of Technology

   Affiliation as printed

   Biology and Bioengineering Department, California Institute of Technology, Pasadena, CA, USA

6. GJ Jensen

   Brigham Young University · California Institute of Technology

   Affiliation as printed

   Biology and Bioengineering Department, California Institute of Technology, Pasadena, CA, USA and Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT USA

7. GP Resch
   Affiliation as printed

   Nexperion e.U. – Solutions for Electron Microscopy, Vienna, Austria

8. Ariane Briegel corresponding Aachen Department of Microbial Sciences Institute of Biology, Leiden University Netherlands Center for Nanoscopy (NeCEN)

   Leiden University

   Affiliation as printed

   Department of Microbial Sciences, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

   Netherlands Center for Nanoscopy (NeCEN), Leiden University, The Netherlands