Cargando…

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

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 high-quality samples has become a bottleneck in the cryo-EM structure-determinatio...

Descripción completa

Detalles Bibliográficos
Autores principales: Hrebík, Dominik, Gondová, Mária, Valentová, Lucie, Füzik, Tibor, Přidal, Antonín, Nováček, Jiří, Plevka, Pavel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9629489/
https://www.ncbi.nlm.nih.gov/pubmed/36322417
http://dx.doi.org/10.1107/S2059798322009299
_version_ 1784823405440139264
author Hrebík, Dominik
Gondová, Mária
Valentová, Lucie
Füzik, Tibor
Přidal, Antonín
Nováček, Jiří
Plevka, Pavel
author_facet Hrebík, Dominik
Gondová, Mária
Valentová, Lucie
Füzik, Tibor
Přidal, Antonín
Nováček, Jiří
Plevka, Pavel
author_sort Hrebík, Dominik
collection PubMed
description 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 high-quality 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 air–water 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.
format Online
Article
Text
id pubmed-9629489
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher International Union of Crystallography
record_format MEDLINE/PubMed
spelling pubmed-96294892022-11-14 Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules Hrebík, Dominik Gondová, Mária Valentová, Lucie Füzik, Tibor Přidal, Antonín Nováček, Jiří Plevka, Pavel Acta Crystallogr D Struct Biol Research Papers 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 high-quality 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 air–water 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. International Union of Crystallography 2022-10-20 /pmc/articles/PMC9629489/ /pubmed/36322417 http://dx.doi.org/10.1107/S2059798322009299 Text en © Dominik Hrebík et al. 2022 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
spellingShingle Research Papers
Hrebík, Dominik
Gondová, Mária
Valentová, Lucie
Füzik, Tibor
Přidal, Antonín
Nováček, Jiří
Plevka, Pavel
Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title_full Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title_fullStr Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title_full_unstemmed Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title_short Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
title_sort polyelectrolyte coating of cryo-em grids improves lateral distribution and prevents aggregation of macromolecules
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9629489/
https://www.ncbi.nlm.nih.gov/pubmed/36322417
http://dx.doi.org/10.1107/S2059798322009299
work_keys_str_mv AT hrebikdominik polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT gondovamaria polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT valentovalucie polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT fuziktibor polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT pridalantonin polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT novacekjiri polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules
AT plevkapavel polyelectrolytecoatingofcryoemgridsimproveslateraldistributionandpreventsaggregationofmacromolecules