Cryo-EM structure of the untreated YiiP/Fab complex (SP1). (A) Density map at 3.8 Å overlaid with a refined atomic model with YiiP (cyan) and Fab molecules (orange and red for the light chain and heavy chain, respectively). (B) Superposition of the refined atomic model from SP1 (cyan) with the previous atomic model for YiiP (PDB accession no. 5VRF, dark blue) showing that both models represent the same inward-facing conformation (RMSD for Cα atoms is 1.5 Å; Table 3). Locations of the individual Zn sites (A, B, and C) are indicated by arrows. (C) Superposition of the SP1 model (cyan, orange, and red) with the x-ray model of YiiP from E. coli (PDB accession no. 3H90, green). Substantial conformational differences in the TMD are evident and reflected in the high RMSD for Cα atoms of 10.3 Å (Table 3). (D–F) Close-up views of density at the individual Zn sites in the SP1 structure. Metal ions are displayed as pink spheres. (G) C_α RMSD plots for one of the three MD simulations (md1) of the holo state shows relatively little change in the dimer over the course of the simulation. The three traces correspond to the different alignment schemes relative to the starting model (5VRF): “All” indicates global alignment based on the entire molecule, “TMD” indicates alignment based on the TMD only, and “CTD” indicates alignment based on CTD only. Analogous plots for md0 and md2 simulations are shown in Fig. S5. (H) Distributions of RMSD derived from all three simulations using the alignment schemes indicated in the legend. These RMSD distributions, as well as those shown in other figures, were generated with a KDE. (I) RMSF plotted for each residue based on the different alignment schemes. RMSF profiles were averaged over both protomers and all three simulations, with the mean shown as the solid line and the error band indicating the SD over these six profiles. The dashed line represents the boundary between TMD and CTD.