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TitlePolyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules.
Journal, issue, pagesActa Crystallogr D Struct Biol, Vol. 78, Issue Pt 11, Page 1337-1346, Year 2022
Publish dateNov 1, 2022
AuthorsDominik Hrebík / Mária Gondová / Lucie Valentová / Tibor Füzik / Antonín Přidal / Jiří Nováček / Pavel Plevka /
PubMed AbstractCryo-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, ...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.
External linksActa Crystallogr D Struct Biol / PubMed:36322417 / PubMed Central
MethodsEM (single particle)
Resolution1.77 - 3.8 Å
Structure data

14679

7ze1

EMDB-14679, PDB-7ze1:
Tribolium castaneum hexamerin 2
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-14680: Tribolium castaneum hexamerin 2 from aggregated sample
Method: EM (single particle) / Resolution: 3.1 Å

14704

7zfw

EMDB-14704, PDB-7zfw:
Structure of human 80S ribosome obtained from ssDNA coated grid
Method: EM (single particle) / Resolution: 3.8 Å

14705

7zg7

EMDB-14705, PDB-7zg7:
Structure of human Apoferritin obtained from ssDNA coated grid
Method: EM (single particle) / Resolution: 1.77 Å

Chemicals

ChemComp-ZN:
Unknown entry

ChemComp-NA:
Unknown entry

ChemComp-HOH:
WATER / Water

Source
  • tribolium castaneum (red flour beetle)
  • red flour beetle (red flour beetle)
  • homo sapiens (human)
KeywordsUNKNOWN FUNCTION / hexamerin / tribolium castaneum / storage protein / RIBOSOME / human 80S ribosome / ssDNA covered grid / ssDNA / cryoEM / METAL BINDING PROTEIN / apoferritin

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