FIG 7.
Negative-stain single-particle EM reconstruction of BG505 SOSIP.664 Env bound to 2G12 Fab2 and soluble 2-domain CD4 (sCD4). (A) Coomassie-stained, nonreducing SDS-PAGE gel analysis of the complex components and the complex purified by size exclusion chromatography. BG505 SOSIP.664, 2G12 Fab, and sCD4 were stained similarly with equal amounts of protein. Note that some 2G12 F(ab′)2 remained with 2G12 Fab2 during purification and subsequently bound to SOSIP; because 2G12 is domain exchanged, these two products are virtually identical except on a nonreducing SDS-PAGE gel. (B) SEC-MALS was used to determine that the observed shift in elution volume and molar mass for the SOSIP-2G12 Fab-sCD4 (MMComplex, red) complex in comparison to those of the unliganded trimer (MMSOSIP, blue) correspond to one trimer binding three Fab2 and three sCD4 molecules simultaneously. (C) Structural characterization of BG505 SOSIP.664 Env bound to three 2G12 Fab2 and three sCD4 by negative-stain single-particle EM. Top (left) and side (right) views of the reconstruction are rendered with the segmented densities corresponding to 2G12 Fab2 in blue and BG505 SOSIP.664 Env in white. (D) Crystal structures of 2G12 Fab2 (PDB ID 1OP5) and gp120 bound to sCD4 (PDB ID 2B4C) (53) were fit into the EM density map. All glycans were removed for clarity. Top (left) and side (right) views of the fitted maps are shown with gp120 rendered in yellow, V3 loops in red, sCD4 in gray, and 2G12 Fab2 in blue.