Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F21%3A00124267" target="_blank" >RIV/00216224:14740/21:00124267 - isvavai.cz</a>
Result on the web
<a href="https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo" target="_blank" >https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.3791/62351" target="_blank" >10.3791/62351</a>

Result language
angličtina
Original language name
Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
Original language description
Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.
Czech name
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Czech description
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Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology

Project
<a href="/en/project/LM2018127" target="_blank" >LM2018127: Czech Infrastructure for Integrative Structural Biology</a><br>
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year
2021
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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