(A) View (stereo) into the selectivity filter of CLC-1. αN, αF, αR, and αC-D segments (Cα trace and side chains) are shown in cyan, salmon, yellow, and olive, respectively. The side chains of Glugate, TyrC, and SerC are represented with balls and sticks. Cl−-binding sites are indicated by green (Sext and Sint) and gray (Scen) spheres. The cryo-EM density map is shown in mesh (Sext and Sint in magenta and the rest in gray). (B) Water-accessible regions in the filter region, probed by Hollow (Ho and Gruswitz, 2008), are shown with purple (extracellular vestibule) and blue (intracellular vestibule) dots. Glugate is represented in ball-and-stick. (C) Comparison of Glugate positions between the CLC-1 channel and CLC transporters. The amino acid segments 146–149 and 355–358 forming the anion selectivity filter were aligned between structures. Cα-traces of the segments are shown with the Glugate side chains in ball-and-stick representation. Blue, CLC-1. Light orange, WT EcCLC (PDB ID: 1OTS). Yellow, EcCLC E148Q mutant (PDB ID: 1OTU). Magenta, CmCLC (PDB ID: 3ORG). Gray spheres represent the positions of Cl− ions seen in EcCLC E148Q mutant (Sext and Scen). Note that the Cl− ion at CLC-1’s Sext (not shown) essentially coincides with Sext of EcCLC.