Zn binding controls a hydrophobic gate on the cytoplasmic side of the TMD. (A – C) Three orthogonal views of the cryo-EM structure of the symmetric holo state (SP3sym) showing clear separation between Leu154 and Leu199. (B) 90° rotation about the vertical axis relative to A. (C) 90° rotation about the horizontal axis relative to B. Helices in the TMD have rainbow colors ranging from blue for M1 to red for M6. (D–F) Analogous views of the cryo-EM structure from the asymmetric apo state (SP2) showing how bending of M5 brings Leu154 and Leu199 close together. (G) Overview of the YiiP dimer showing the locations of residues used as collective variables: Leu154 and Leu199 are blue, whereas Ala43 and Ala185 on the periplasmic side of the membrane are red. Membrane helices have the same rainbow colors. (H) Distances between C_α atoms of Ala43 and Ala185 (D_peri) for each protomer during the three simulations in the apo (purple) and holo (cyan) states. Distributions calculated as KDEs are shown on the right. The corresponding values of D_peri from the SP3sym and SP2 structures are shown as dashed horizontal lines; asymmetry in SP2 produces different values for each monomer. (I) Distances between C_α atoms of Leu154 and Leu199 (D_cyto) for simulations of the apo and holo states with distributions shown on the right. D_cyto from the SP3sym and SP2 structures are shown as horizontal dashed lines.