Fig. 3: Cryo-EM structures of CLC-ec1.

a–c Cryo-EM density maps for CLC-ec1, with each panel showing one of the two subunits in color: pH 7.5, orange; pH 4.0, purple; pH 3.0, cyan. d–f Molecular models of CLC-ec1 for structure determination at pH 7.5, 4.0, and 3.0, with colors as in (a–c). The zoomed-in views of the models showing key residues and resolved Cl⁻ ions. Cartoons depictions to the right of the zoomed-in views are as in Fig. 1, highlighting the E_gate conformations observed at pH 7.5 (“middle” conformation), pH 4 (“middle” and “out” conformations), and pH 3 (“out” conformation). g View from the intracellular side, highlighting major conformational changes between pH 7.5 and 4.0. Residues lining the H⁺ pathway are colored in green (E113, E148, A182, L186, A189, F190, F199, E202, E203, M204, I402, T407, and Y445), to highlight the opening of this pathway in the pH 4.0 conformation. h Comparison of pH 4.0 and pH 3.0 cryo-EM structures to the QQQ crystal structure. The structural alignment was done in ChimeraX using the matchmaker command, aligning the dimer. The high RMSD at the H–I loop reflects greater flexibility in the pH 3.0 structure. At pH 7.0 (QQQ), Q207 interacts with E117, stabilizing the loop. Protonation of E117 at pH 3.0 likely disrupts this interaction, increasing loop mobility. i Pore radius profiles calculated using HOLE. j Zoomed-in view comparing E148 and F357 between pH 4 (out) and pH 3. The inner gate residue Y445 is shown for reference.