UVC inactivation of pathogenic samples suitable for cryo-EM analysis.

Area of Science:

• Structural Biology
• Microbiology
• Biophysics

Background:

• Cryo-electron microscopy (cryo-EM) is vital for studying biological structures, especially pathogens.
• Pathogen research is limited by biosafety concerns, often requiring chemical fixation that alters ultrastructure.
• Alternative non-pathogenic models lack the complexity of target pathogens.

Purpose of the Study:

• To investigate ultraviolet-C (UVC) radiation as a method to inactivate pathogens for cryo-EM.
• To assess the impact of UVC treatment on sample ultrastructure using cryo-EM techniques.
• To validate UVC treatment efficacy on a standard protein sample for high-resolution cryo-EM.

Main Methods:

• Application of UVC radiation at cryogenic temperatures to Vibrio cholerae and ICP1 bacteriophage.
• Cryo-electron tomography (cryo-ET) with sub-tomogram averaging to analyze structural integrity.
• Single particle analysis (SPA) to evaluate structural preservation and resolution.
• UVC treatment and cryo-EM SPA of apoferritin (ApoF) as a control sample.

Main Results:

• UVC treatment significantly reduced or eliminated infectivity of Vibrio cholerae and ICP1 bacteriophage.
• No discernible structural damage was observed in treated pathogens via cryo-ET and SPA.
• Cryo-EM SPA of UVC-treated apoferritin yielded a high-resolution structure (2.1 Å) indistinguishable from untreated samples.
• UVC irradiation did not compromise the ultrastructure or data quality for high-resolution cryo-EM.

Conclusions:

• UVC radiation at cryogenic temperatures is an effective, inexpensive method for pathogen inactivation in cryo-EM sample preparation.
• This technique preserves sample ultrastructure, overcoming limitations of chemical fixation and non-pathogenic models.
• UVC treatment is a valuable addition to the cryo-EM toolbox, enabling safer and more accurate structural studies of infectious agents.