X-ray crystal structures of CLC-ec1. CLC-ec1 mutants E203H (PDB accession no. 3EJY) and E203V (3EJZ) were crystallized with Br⁻ substituting for Cl⁻ in all solutions. Structures are displayed with the extracellular side facing upward. (A) Structural alignment of CLC variants. Backbone trace of homodimeric WT protein is shown in gray, with single subunits of E203H (yellow) and E203V (cyan) superimposed by alignment of residues 20–200. Blue spheres represent Cl⁻ ions, and Glu_in is indicated by space-filled atoms in the WT. (B) Stereo views of ion-coupling region between Glu_in and Glu_ex positions. Br⁻ ions appear as anomalous difference maps (red) contoured at 5σ. 2F_o–F_c electron density maps (black) are shown near the Glu_in position, including R28, contoured at 0.8–1 σ. Outer gate (Glu_ex) and inner gate (S107/Y445) are shown in green sticks. Part of the N-terminal helix from the twin subunit carrying R28 is shown in blue; Val and His substitutions at Glu_in and R28 are rendered in yellow/blue sticks.