(A) Refined 3D reconstructions from negative stain EMPEM. Specificities are mapped onto the Env trimer structure, with epitopes colored as in (C). (B) Mutational antigenic profiling data mapped onto the BG505 SOSIP Env structure, as in Figure 2C, represented here to contrast with EMPEM. (C) Summary data from (A) and (B), as well as validation TZM-bl neutralization assay point-mutant mapping of single and double epitope knock out mutations (see Figure 3—figure supplement 1 for details). A checkmark (EMPEM and MAP) indicates a response was detected to that epitope, and TZM-bl responses were summarized as ‘Dominant’, ‘Subdominant’, or undetected (left blank) based on data in Figure 3—figure supplement 1. We did not perform neutralization assay validation at epitopes where the discrepancies between the EMPEM and mutational antigenic profiling are easily explained by antigenicity differences between soluble trimer and virus (N611, base, and gp120 interface; labeled as ‘Not tested’).