Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules
| Autoři | |
|---|---|
| Rok publikování | 2022 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Acta Crystallographica Section D: Structural Biology |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://journals.iucr.org/d/issues/2022/11/00/jb5048/index.html |
| Doi | http://dx.doi.org/10.1107/S2059798322009299 |
| Klíčová slova | cryo-EM; sample preparation; ssDNA; coating cryo-EM grids; aggregation |
| Přiložené soubory | |
| Popis | 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. |
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