From Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an Example

Diebolder, Christoph A.; Dillard, Rebecca S.; Renault, Ludovic

doi:10.1007/978-1-0716-1406-8_12

Cited by 4 publications

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References 60 publications

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“…Apoferritin samples were treated as described in Diebolder et al 26 . Briefly, grids were loaded into a Titan Krios electron microscope (TFS) operated at 300 kV, equipped with a Gatan K3 BioQuantum direct electron detector (Gatan, Inc).…”

Section: Methodsmentioning

confidence: 99%

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

et al. 2022

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Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. 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 pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the 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 applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, 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 indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

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Section: Methodsmentioning

confidence: 99%

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

et al. 2022

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“…Indeed, apoF has been used as such kind of model for testing TEM performances [27] [25]. Its molecular weight is ∼ 500 kDa and is composed of 24 subunits arranged in an octahedral symmetry [23].…”

Section: First Experiments With the Model Protein Apoferritinmentioning

confidence: 99%

Improving electron microscopic hardware to push the resolution limits in single particle cryo-EM

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UVC inactivation of pathogenic samples suitable for cryoEM analysis

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Renault

Althof

et al. 2021

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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 A 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.

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From Tube to Structure: SPA Cryo-EM Workflow Using Apoferritin as an Example

Cited by 4 publications

References 60 publications

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

Improving electron microscopic hardware to push the resolution limits in single particle cryo-EM

UVC inactivation of pathogenic samples suitable for cryoEM analysis

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