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[Preprint]. 2024 Oct 24:2024.10.23.619923. [Version 1] doi: 10.1101/2024.10.23.619923

Figure 2. Design and validation of oP3h and I5350A2hN trimers for prefusion gB stabilization.

Figure 2.

(a) Wheel diagram (top) and AlphaFold2-predicted structure of the oP3h trimer, shown in ribbon representation with residues labeled (bottom). (b) Top, Coomassie stained SDS-PAGE of SARS-CoV-2 S ectodomains with C-terminally fused foldon, one to six oP3h (RINEIER) repeats, or with four oP3h (RINAIET) repeats. Glycoproteins were expressed and purified in parallel and normalized to a standard volume for comparison of relative yields after affinity purification. Bottom, the proportion of S ectodomains (after affinity purification) that are clearly folded in the prefusion state, as determined by negative stain 2D classification. Exemplar prefusion 2D class averages are shown underneath. (c) CryoEM maps of SARS-CoV-2 S-oP3h with three RINEIER (top) or four RINAIET (bottom) C-terminal repeats with each protomer colored distinctly. Insets show local refinement maps (mesh) and refined models (ribbons) encompassing the oP3h fusion. (d) Top, Coomassie stained non-reducing SDS-PAGE analysis of EBV gB deliD with (+) and without (−) oP3h. Bottom, cryoEM map (mesh) and model (ribbons) of the EBV gB deliDoP-I5350A2h domain V (DV) and oP3h fusion. (e) Design and AlphaFold2-predicted models of I5350A2h and I5350A2hN, showing the predicted relative orientation of DI to which they are fused. (f) Left, Coomassie stained non-reducing SDS-PAGE and 2D EM class averages of negatively stained gB-deliD ectodomains with (+) and without (−) oP3h, A293P (P), or I5350A2h. (g) Top, Schematic showing the addition of 1 (center) or 4 (right) heptad repeats to the coiled coils of I5350A2h. Bottom, EM reconstructions of negatively stained EBV gB deliDoP-I5350A2h with heptad repeats added to the coiled coils of I5350A2h. (h) Local refinement cryoEM maps (transparent surface) and model (black ribbons) of the I5350A2h or I5350A2hN regions within EBV gB deliDoP-I5350A2h and EBV gB deliDoP-I5350A2hN, respectively; blue arrows, the relative orientation of DI. The diameter at which DI is held by I5350A2h or I5350A2hN is labeled on the right.