A fine-grained view of two clusters of shifted sites, both of which are in conformationally dynamic regions of Env. In each structure, we only show side chains for shifted sites or other sites of interest, which include substituted sites and sites that are proposed to take part in a hydrophobic network that helps control Env’s dynamics (Ozorowski et al., 2017). These side chains are colored according to the key in the upper right. (A) A cluster of four shifted sites at the apex of the closed pre-fusion conformation of Env (Stewart-Jones et al., 2016), a region that is not resolved in the open CD4-bound conformation. These sites are in Env’s first/second variable loop (164 and 165) and its third variable loop (307 and 309), which pack against one another and against an adjacent Env protomer (the inter-protomer boundary is indicated by a dashed line). Each of the shifted sites has substituted. And these sites are in the immediate vicinity of other non-shifted, but substituted sites. Thus, it seems likely that these shifts at least partially arise from short-range epistatic interactions within this cluster. However, longer range epistatic interactions with more distant sites also seem plausible given the highly dynamic nature of this region. (B) A cluster of shifted sites that are adjacent to a network of hydrophobic residues (blue/purple) that help mediate the conformational change between the closed pre-fusion state to the open CD4-bound state (Ozorowski et al., 2017). One of the shifted sites (purple) is in this network. These sites are shown in context of both structural states, the latter of which is from Ozorowski et al., 2017). Nearly one third of the shifted residues cluster within this structural region (9/30). In contrast to panel (A), only a few of the shifted sites are adjacent to substitutions or have substituted themselves. This trend is found in both the closed and open conformations, suggesting that the shifts may be primarily due to long-range epistatic interactions.