Fig. 1: Structural determination of the PEDV PT52 S by cryo-ET and STA.
From: In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM
a Workflow of initial template generation for the S protein. Positions and orientations of S proteins were manually defined as indicated by fuchsia circles and cyan arrows, respectively, to extract the subtomograms. Iterations of STA were performed to generate the initial template. Indicated by hot pink circles and cyan arrows, respectively, to extract the subtomograms. Iterations of STA were performed to generate the initial template. b Workflow of subtomogram analysis. Membrane coordinates were approximated by a dashed red circle to define the center (x, y, z) and radius (r) of the viral particle. The membrane patches were aligned and colored green-to-red corresponding to high-to-low cross-correlation (CC) as indicated by the color scale bar on the left. The membrane coordinates were shifted outward from the center to help identify spikes. Duplicated spike coordinates and dust were removed before further analyses. c Symmetry expansion and mask definition for PCA analysis. Each S protein subtomograms were sub-boxed (dashed purple rectangle) to triplicate particles by symmetry expansion. A spherical PCA mask colored in purple was defined to approximate the location where D0 heterogeneity was expected. d Scatter plot of PC2 and PC3 of the PCA to reveal the D0 heterogeneity (left panel). Projection along PC3 was divided into three subclasses corresponding to D0 down, D0 up, and their mixture, i.e., D0 mix (right panel). e Averaged D0 conformation models (up, down, and mix) and a noise volume were used as reference templates in the multi-reference alignment task to classify all the protomer subtomograms. Classified protomers were mapped back to the original S trimers for further refinement. f Our cryo-ET results of PEDV PT52 S structures revealed that S protein with four different D0 conformational arrangements in the pre-fusion state and one in the post-fusion state. The pie chart illustrates the respective populations which include D0DDD (hot pink), D0UDD (blue), D0UUD (purple), and D0UUU (yellow). Orthogonal views of the reconstructed postfusion S structure in a semi-transparent orange surface superimposed with a homology model.
