Polyelectrolyte coating of cryo-EM grids improves lateral
distribution and prevents aggregation of macromolecules

J 2022

Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules

HREBÍK, Dominik; Mária GONDOVÁ; Lucie VALENTOVÁ; Tibor FÜZIK; Antonin PRIDAL et al.

Základní údaje

Originální název

Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules

Autoři

HREBÍK, Dominik; Mária GONDOVÁ; Lucie VALENTOVÁ ; Tibor FÜZIK ; Antonin PRIDAL; Jiří NOVÁČEK a Pavel PLEVKA

Vydání

Acta Crystallographica Section D: Structural Biology, Chester, International Union of Crystallography, 2022, 2059-7983

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 2.200

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14740/22:00128514

Organizační jednotka

Středoevropský technologický institut

Klíčová slova anglicky

cryo-EM; sample preparation; ssDNA; coating cryo-EM grids; aggregation

Štítky

CF CRYO, NIVB_CEITEC, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 27. 10. 2024 14:23, Ing. Martina Blahová

Anotace

V originále

Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of highquality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the airwater interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.

Návaznosti

GX19-25982X, projekt VaV

Název: Analýza replikace enterovirů s využitím elektronové mikroskopie

Investor: Grantová agentura ČR, Structural study of enterovirus replication in situ

LX22NPO5103, projekt VaV

Název: Národní institut virologie a bakteriologie (Akronym: NIVB)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Národní institut virologie a bakteriologie, 5.1 EXCELES

90127, velká výzkumná infrastruktura

Název: CIISB II

Přiložené soubory

jb5048.pdf

Citovat

HREBÍK, Dominik; Mária GONDOVÁ; Lucie VALENTOVÁ; Tibor FÜZIK; Antonin PRIDAL; Jiří NOVÁČEK a Pavel PLEVKA. Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules. Acta Crystallographica Section D: Structural Biology. Chester: International Union of Crystallography, 2022, roč. 78, NOV, s. 1337-1346. ISSN 2059-7983. Dostupné z: https://doi.org/10.1107/S2059798322009299.

@article{2252344,
   author = {Hrebík, Dominik and Gondová, Mária and Valentová, Lucie and Füzik, Tibor and Pridal, Antonin and Nováček, Jiří and Plevka, Pavel},
   article_location = {Chester},
   article_number = {NOV},
   doi = {https://doi.org/10.1107/S2059798322009299},
   keywords = {cryo-EM; sample preparation; ssDNA; coating cryo-EM grids; aggregation},
   language = {eng},
   issn = {2059-7983},
   journal = {Acta Crystallographica Section D: Structural Biology},
   title = {Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules},
   url = {https://journals.iucr.org/d/issues/2022/11/00/jb5048/index.html},
   volume = {78},
   year = {2022}
}

TY  - JOUR
ID  - 2252344
AU  - Hrebík, Dominik - Gondová, Mária - Valentová, Lucie - Füzik, Tibor - Pridal, Antonin - Nováček, Jiří - Plevka, Pavel
PY  - 2022
TI  - Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
JF  - Acta Crystallographica Section D: Structural Biology
VL  - 78
IS  - NOV
SP  - 1337-1346
EP  - 1337-1346
PB  - International Union of Crystallography
SN  - 20597983
KW  - cryo-EM
KW  - sample preparation
KW  - ssDNA
KW  - coating cryo-EM grids
KW  - aggregation
UR  - https://journals.iucr.org/d/issues/2022/11/00/jb5048/index.html
N2  - Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of highquality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the airwater interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.
ER  -

HREBÍK, Dominik; Mária GONDOVÁ; Lucie VALENTOVÁ; Tibor FÜZIK; Antonin PRIDAL; Jiří NOVÁČEK a Pavel PLEVKA. Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules. \textit{Acta Crystallographica Section D: Structural Biology}. Chester: International Union of Crystallography, 2022, roč.~78, NOV, s.~1337-1346. ISSN~2059-7983. Dostupné z: https://doi.org/10.1107/S2059798322009299.

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