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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F22%3A00128514" target="_blank" >RIV/00216224:14740/22:00128514 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/62156489:43210/22:43922198

Výsledek na webu

<a href="https://journals.iucr.org/d/issues/2022/11/00/jb5048/index.html" target="_blank" >https://journals.iucr.org/d/issues/2022/11/00/jb5048/index.html</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1107/S2059798322009299" target="_blank" >10.1107/S2059798322009299</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Acta Crystallographica Section D: Structural Biology

ISSN

2059-7983

e-ISSN

—

Svazek periodika

78

Číslo periodika v rámci svazku

NOV

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

10

Strana od-do

1337-1346

Kód UT WoS článku

000885880300005

EID výsledku v databázi Scopus

2-s2.0-85141889420