Fig. 5: Spatial distribution and angular orientation analysis with single-particle data.
From: Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects
a, z height distribution of particles in a single micrograph. z height is the distance of particles from the fitted plane, which corresponds to spatial distributions along the z axis. The peak of the KDE fitting curve indicates the mode of z heights, while the zero point corresponds to the mean distance. The unimodal distributions of z heights of apo-ferritin, ACE2 and streptavidin particles are distributed in asymmetrical shapes with a long tail when supported on the holey gold grid with two interfaces in contact with the air (purple), or the EM grids with one interface in contact with the air and the other in contact with the supporting film (green), respectively. In some micrographs, z heights of apo-ferritin particles prepared on rGO show bimodal distributions, indicating adsorption at both AWIs and GWIs. 70S ribosome, 20S proteasome, apo-ferritin, ACE2 and streptavidin particles (blue) show Gaussian-like symmetrical distributions of z heights at the main peak when prepared using the ESI-cryoPrep method. These distributions appear to have the same mean, mode and median, and low absolute skewness value. b, Skewness distribution of particles’ z height across the entire SPA dataset. c, Skewness classification of particles’ z height across the entire SPA dataset. The control particle z height distributions (apo-ferritin without or with supporting films, ACE2 and streptavidin with supporting films from conventional methods) tend to be asymmetric with larger absolute skewness values, while ESI-cryoPrep distribution appears to be more symmetric with close-to-zero skewness values. The color code is compatible with that in a. For each sample skewness distribution, n = 2,821, 3,275, 2,209, 280, 1,606, 1,801, 1,168, 1,793 and 827 independent micrographs were analyzed, progressing from left to right. The center dot of the inner boxplot represents the median, while the box’s limits denote data values within the interquartile range (IQR). Whiskers extend from the box to the furthest data point within 1.5 times the IQR. Data points beyond the whiskers are identified as outliers and are excluded from the primary figure. d, Heatmap of angular orientations of the five protein samples, 70S ribosome (first panel), 20S proteasome (second panel), apo-ferritin (third panel), ACE2 (fourth panel) and streptavidin (fifth panel). Data using the ESI-cryoPrep method are shown on the top panel, while the control data on supporting films using the conventional method are shown on the bottom. AngleRot, AngleTilt are two given Euler angles calculated in RELION.
