Journal: Acta Crystallogr D Struct Biol / Year: 2022 Title: Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules. Authors: Dominik Hrebík / Mária Gondová / Lucie Valentová / Tibor Füzik / Antonín Přidal / Jiří Nováček / Pavel Plevka / Abstract: 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, ...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.
Average exposure time: 5 sec. / Electron dose: 57 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1711
EM imaging optics
Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3106 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
B value: 45.57 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: corelation coefficient / Details: real space refine
Refine LS restraints
Refine-ID
Type
Dev ideal
Number
ELECTRONMICROSCOPY
f_bond_d
0.003
5917
ELECTRONMICROSCOPY
f_angle_d
0.492
8045
ELECTRONMICROSCOPY
f_dihedral_angle_d
4.235
777
ELECTRONMICROSCOPY
f_chiral_restr
0.045
743
ELECTRONMICROSCOPY
f_plane_restr
0.004
1039
+
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