Fo − Fo difference maps reveal local conformational shifts connecting the active site, interdomain interface, and dimer interface. Center: overview of the isomorphous Fo − Fo difference electron-density map at ±3σ (green–red mesh) for the 240 K data set (cyan) minus the 100 K data set (dark blue) (see Fig. S10 for Fo − Fo maps for all temperatures). Ligands from cocrystal structures are shown at the active site (dashed oval) (pale orange, 6lu7), interdomain interface (purple, 5ree; yellow, 5rec), and dimer interface (orange, 7lfp; pink, 5rf0). (a) Glu290 switches from one side-chain rotamer at 100 K to two alternate rotamers at 240 K. Glu290 is spatially adjacent to Cys128, which switches from two alternate rotamers at 100 and 240 K to a single rotamer at 277 K and above in our multiconformer models; the alternate rotamer occupancy is lower at 240 K, consistent with its positive Fo − Fo peak (see Fig. S12). These residues are near two ligands from separate crystallographic screens (7lfp and 5rf0), as well as many ordered PEG molecules from the crystallization cocktails of various structures (7kvr, 7kvl, 7kfi, and 7lfe). (b) An ∼45°-rotated view relative to part (a) shows that these two ligands bind at the dimer interface of the biological monomer, constituted in the crystal from a symmetry-related protomer (gray surface). This interface also includes the Asp197 region (right). (c) Thr198 switches from two alternate side-chain rotamers at 100 K to a single rotamer at 240 K, while Glu240 – located across the interdomain interface – changes side-chain rotamer (curved arrows), with additional effects on the adjacent backbone of Pro241. In the other direction from Asp197 (down in this view), other residues in the P5 substrate-binding pocket loop (Fig. 3) undergo conformational adjustments en route to the active site. Meanwhile, an interacting water molecule at 100 K (blue sphere) becomes displaced at 240 K, and is correspondingly absent in that model.