Preparation and Cryo-FIB micromachining of &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt; for Cryo-Electron Tomography

Moravcová, Jana; Pinkas, Matyáš; Holbová, Radka; Nováček, Jiří

doi:10.3791/62351-v

Cited by 1 publication

(1 citation statement)

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“…20 x 0.2 µm² stress-relief trenches were made with a current of 1.5 nA (Wolff et al, 2019). To remove ice contamination or re-deposition that occurred during the milling procedure, a polishing step was performed (Moravcová et al, 2021). To our experience, polishing should be performed less than 2-3 h before the lamellae are retrieved from the SEM chamber in order to limit re-deposition and fully benefit from the fine milling.…”

Section: Cryo-fibm/sem Workflowmentioning

confidence: 99%

Ultrastructural details of resistance factors of the bacterial spore revealed by in situ cryo-electron tomography

Bauda

Gallet

Moravcová

et al. 2023

Preprint

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The bacterial spore owes its incredible resistance capacities to various molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography (cryo-ET) on bacteria lamellae generated by cryo-focused ion beam micromachining (cryo-FIBM) provides insights into the ultrastructural organization of Bacillus subtilis sporangia, including that of the DNA and nascent coat layers. Analysis of the reconstructed tomograms reveal that rather early during sporulation, the chromosome in the developing spore (the forespore) adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a complex stack of amorphous or structured layers with distinct electron density, dimensions and organization. We investigated the nature of the nascent coat layers in various mutant strains using cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE.

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Section: Cryo-fibm/sem Workflowmentioning

confidence: 99%