Figure 4. Design and characterization of HR1-redesigned Env trimers with short cleavage site linkers.

(a) Schematic representation of CST redesigns and near-full-length (CSF) linkers. (b) SEC profiles of five CST trimers (obtained from ensemble-based de novo protein design, upper panel) and two CSF trimers (lower panel) from a Superdex 200 10/300 column. The UV value of the trimer peak and the percentages of UV values for aggregate peak (at 9 ml) and dimer/monomer peak (at 12 ml) relative to the trimer peak (at 10.5 ml) are labelled for comparison. (c) BN-PAGE of 293F-expressed, GNL-purified CSF trimers following SEC separation on a Superdex 200 16/600 column. The fractions used for EM analysis and antigenic profiling are circled by black dotted lines with expected positions for trimer and dimer bands labelled on the gel. New Env species observed for the CSF trimer are also labelled. (d) 3D reconstructions of CSF (states 1 and 2) and CSF-SOS trimers in unliganded and PGV04-bound forms derived from negative-stain EM. The trimer densities are shown in grey transparent surface with the fitted high-resolution cryo-EM structure of the SOSIP trimer (PDB 3J5M, gp120 in blue with V1V2 in magenta, V3 in green, gp41 in brown and PGV04 Fab in red). Both top and side views are shown after fitting the previously published EM model (PDB ID: 3J5M) into the density.