Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality

Abstract

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 Å, while the resolution of the map reached 4.0 Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4 Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 Å, a marked improvement over the value of 3.9 Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

Protein denaturation at the air-water interface and how to prevent it

“…The standard method of preparing cryo-EM specimens by plunge-freezing of thin, unsupported layers of protein solution is therefore potentially problematic. Recent studies (Glaeser, 2018; Glaeser and Han, 2017; Han et al, 2017) have drawn attention to the effects of the air water interface on proteins in solution, in particular on their integrity and orientation on cryoEM grids.…”

Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction

“…The lattice tilts we identify may ultimately represent the result of various physical phenomena: they may be intrinsic properties of the crystals themselves, limited to only a subset of macromolecular crystals and affected by or result from sample preparation procedures. Current methods in cryoEM sample preparation, for example, may exert forces potent enough to distort crystal lattices and affect molecular structures 67 . Lastly, by allowing nanoscale (20 nm) interrogation of lattice structure in macromolecular crystals, 4DSTEM offers a potential way to shrink the number of molecules required for structure determination by electron diffraction from single or serial crystal data.…”

Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution

“…But these may all unavoidably reduce the contrast of the molecules in cryo-EM. An ultimate solution to prevent macromolecules from hitting the AWI is by blocking the AWI with either a supporting film such as the graphene or some inert surfactant that does not has any impact on the macromolecules' structures as suggested by Glaeser 10,34,35 . Anchoring the macromolecules with certain affinity tags to the graphene or other electron-transparent supporting materials would be a possible solution.…”