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. 2020 Dec 26;34(2):108630. doi: 10.1016/j.celrep.2020.108630

Figure 2.

Figure 2

Biophysics, Antigenicity, and Structure of the S-GSAS Ectodomain in Relation to S-GSAS/PP

(A) Size-exclusion chromatography (SEC) elution profile on a Superose 6 10/300 column of the S-GSAS/PP (black) and S-GSAS (red) ectodomains. Fractions isolated for further characterization are indicated by vertical red dotted lines. Elution volumes of molecular weight standards at 669 (thyroglobulin) and 44 kDa (ovalbumin) are labeled for reference.

(B) SDS-PAGE of the SEC purified ectodomains.

(C) Representative NSEM micrograph of S-GSAS and 2D class averages (related to Data S1).

(D) Binding of ACE2 receptor ectodomain (RBD-directed), CR3022 (RBD-directed neutralizing antibody), 2G12 (S2-directed), Ab712199 (RBD-directed neutralizing antibody), and Ab511584 (S2-directed non-neutralizing antibody) to S-GSAS (red) and S-GSAS/PP (black) measured by ELISA. The schematic shows the assay format. Serially diluted S protein was bound in individual wells of 384-well plates, which were previously coated with streptavidin. Proteins were incubated and washed; then antibodies at 10 μg/mL or ACE2 with a mouse Fc tag at 2 μg/mL were added. Antibodies were incubated and washed, and binding was detected with goat anti-human horseradish peroxidase (HRP).

(E) Differential scanning fluorimetry (DSF) of the S-GSAS (red) and S-GSAS/PP (black) S ectodomains. Thermal melting inflection points (Ti) are indicated on the first derivative graph and reported in the table below from a triplicate.

(F) Side and top view of the cryo-EM reconstructions of the 1-RBD-up (EMD:22822) and the 3-RBD-down (EMD:22821) states of the S-GSAS ectodomain colored by chain. The up positioned RBD in the map is identified by an asterisk (related to Table S1 and Data S1).

(G) Superposition of the 1-up (left; PDB: 7KDH and 6VYB) and 3-down (right; PDB: 7KDG and 6VXX) structures of S-GSAS (red) and S-GSAS/PP (green). All Cα atoms were used for the superpositions.

(H) Magnified view of one protomer from the 1-RBD-up model showing residues K986 and V987 from S-GSAS (colored according to F, overlaid with S-GSAS/PP; PDB: 6VYB; yellow), showing residues P986 and P987 in sticks (related to Figure S1).