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

J 2021

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

MORAVCOVÁ, Jana, Matyáš PINKAS, Radka HOLBOVÁ a Jiří NOVÁČEK

Základní údaje

Originální název

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

Autoři

MORAVCOVÁ, Jana (203 Česká republika, domácí), Matyáš PINKAS (203 Česká republika, domácí), Radka HOLBOVÁ (203 Česká republika, domácí) a Jiří NOVÁČEK (203 Česká republika, garant, domácí)

Vydání

Jove-Journal of Visualized Experiments, CAMBRIDGE, JOURNAL OF VISUALIZED EXPERIMENTS, 2021, 1940-087X

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 1.424

Kód RIV

RIV/00216224:14740/21:00124267

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.3791/62351

UT WoS

000726748200034

Klíčová slova anglicky

VITREOUS SECTIONS; IMPLEMENTATION; CELLS

Štítky

CF CRYO, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 18. 10. 2024 09:34, Ing. Jana Kuchtová

Anotace

V originále

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.

Návaznosti

LM2018127, projekt VaV

Název: Česká infrastruktura pro integrativní strukturní biologii (Akronym: CIISB)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Czech Infrastructure for Integrative Structural Biology

90127, velká výzkumná infrastruktura

Název: CIISB II

Citovat

MORAVCOVÁ, Jana, Matyáš PINKAS, Radka HOLBOVÁ a Jiří NOVÁČEK. Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography. Jove-Journal of Visualized Experiments. CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS, 2021, NOV, č. 177, s. „e62351“, 16 s. ISSN 1940-087X. Dostupné z: https://dx.doi.org/10.3791/62351.

@article{1836561,
   author = {Moravcová, Jana and Pinkas, Matyáš and Holbová, Radka and Nováček, Jiří},
   article_location = {CAMBRIDGE},
   article_number = {177},
   doi = {http://dx.doi.org/10.3791/62351},
   keywords = {VITREOUS SECTIONS; IMPLEMENTATION; CELLS},
   language = {eng},
   issn = {1940-087X},
   journal = {Jove-Journal of Visualized Experiments},
   title = {Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography},
   url = {https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo},
   volume = {NOV},
   year = {2021}
}

TY  - JOUR
ID  - 1836561
AU  - Moravcová, Jana - Pinkas, Matyáš - Holbová, Radka - Nováček, Jiří
PY  - 2021
TI  - Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
JF  - Jove-Journal of Visualized Experiments
VL  - NOV
IS  - 177
SP  - „e62351“
EP  - „e62351“
PB  - JOURNAL OF VISUALIZED EXPERIMENTS
SN  - 1940087X
KW  - VITREOUS SECTIONS
KW  - IMPLEMENTATION
KW  - CELLS
UR  - https://www.jove.com/pdf/62351/jove-protocol-62351-preparation-cryo-fib-micromachining-saccharomyces-cerevisiae-for-cryo
N2  - 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.
ER  -

MORAVCOVÁ, Jana, Matyáš PINKAS, Radka HOLBOVÁ a Jiří NOVÁČEK. Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography. \textit{Jove-Journal of Visualized Experiments}. CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS, 2021, NOV, č.~177, s.~„e62351“, 16 s. ISSN~1940-087X. Dostupné z: https://dx.doi.org/10.3791/62351.

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