Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without...

Area of Science:

• Structural Biology
• Biochemistry
• Cryo-Electron Microscopy

Background:

• Single-particle cryo-electron microscopy (cryo-EM) is crucial for determining enzyme-nucleosome complex structures.
• Cryo-EM sample preparation is hindered by denaturation at the air-water interface (AWI), leading to inconsistent results.
• Existing methods often require crosslinking, which can introduce artifacts.

Purpose of the Study:

• To develop improved cryo-EM grids for better sample preparation of enzyme-nucleosome complexes.
• To overcome air-water interface (AWI) denaturation challenges in cryo-EM.
• To enable high-resolution structural determination of the SNF2h-nucleosome complex without crosslinking.

Main Methods:

• Development of graphene-oxide-coated EM grids functionalized with single-stranded DNA (ssDNA) or a thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer.
• Application of these functionalized grids to protect SNF2h-nucleosome complexes from AWI denaturation during sample preparation.
• Collection of high-quality cryo-EM micrographs and subsequent 3D variability analysis.

Main Results:

• The novel grids successfully protected SNF2h-nucleosome complexes, yielding high-quality micrographs.
• Cryo-EM data allowed for the determination of structural maps ranging from 2.3 to 3 Å resolution.
• 3D variability analysis revealed nucleotide-state-dependent conformational changes and asymmetric protomer coordination within the SNF2h-nucleosome complex.

Conclusions:

• Functionalized graphene-oxide EM grids effectively mitigate AWI denaturation, facilitating high-resolution cryo-EM of enzyme-nucleosome complexes.
• The developed method enables structural studies of intact complexes without crosslinking, providing insights into SNF2h dynamics.
• This approach is broadly applicable to other enzyme-nucleosome and protein-nucleic acid complex structural analyses.