Fig. 2: Demonstration of ice thickness of specimens prepared with ESI-cryoPrep.
From: Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects
a–c, Micrographs of four cryo-specimens, 70S ribosome, 20S proteasome, apo-ferritin and ACE2, were screened at low (×60 or ×130) (a), medium (×320 or ×740) (b) and imaged at high (×29,000 or ×105,000) (c) magnifications under optimal conditions. Orange squares indicate areas with suitable contrast for data collection, while purple squares indicate areas with opaque contrast due to thick ice. The ice thickness distribution is highest in the middle and decreases in a wedge shape toward the edges, providing suitable thin ice for macromolecules of various sizes. Micrograph fast Fourier transforms (b) reveal thin ice thickness, robust particle signals and distinct graphene diffraction patterns. d, Ice thickness measurements were obtained from 3D particle distributions in both single-particle datasets and tomograms for the four tested proteins. Ice thickness in the single-particle data is on the same scale as the monolayer particle distribution in the tomograms. n = 1,426, 3 and 6, n = 1,720 and 8, n = 1,038 and 8, n = 1,768 and 18 independent micrographs and tomograms were analyzed for each macromolecule specimen. The central measure of the error bar is the mean, while the error bar represents the 95% confidence interval. Scale bars, 200 μm (a), 40 μm (b) and 40 nm (c).
