CLC-ec1 and variants were expressed in E. coli, purified, and crystallized after complexation with a FAB fragment of antibody 10EC3/G4 (National Cell Culture Center), as previously described (Dutzler et al., 2003 (link)). Crystals were harvested after 1–3 weeks at 20°C and were frozen in liquid nitrogen. Crystallographic datasets were collected at the Swiss Light Source or the Advanced Photon Source, using radiation at the Br absorption edge (0.919 Å). Diffraction images were indexed and integrated in the HKL program, and electron density and anomalous difference maps were calculated in the CCP4 suite, after molecular replacement with PHASER and refinement with REFMAC5, using the wild-type CLC-ec1-FAB model (accession no. 1OTS) and twofold NCS restraints for the CLC homodimer. The refined model was also minimally rebuilt using sigmaa-weighted 2Fo-Fc and Fo-Fc maps in the COOT program. Model bias was suppressed by prime-and-switch density modification in the SOLVE/RESOLVE program within the PHENIX suite (Terwilliger, 2000 (link)).
CLC-ec1 ion transport functions were assayed by 36Cl− and H+ fluxes in liposomes and voltage-clamped currents in planar lipid bilayers as previously described (Accardi et al., 2004 (link); Accardi and Miller, 2004 (link)); in preparations used for lipid bilayer experiments, an anion-exchange chromatography step was used in lieu of gel filtration to remove outer membrane porins (Accardi and Miller, 2004 (link)). Figures were produced in PyMOL Molecular Graphics (DeLano Scientific,http://www.pymol.org ).
CLC-ec1 ion transport functions were assayed by 36Cl− and H+ fluxes in liposomes and voltage-clamped currents in planar lipid bilayers as previously described (Accardi et al., 2004 (link); Accardi and Miller, 2004 (link)); in preparations used for lipid bilayer experiments, an anion-exchange chromatography step was used in lieu of gel filtration to remove outer membrane porins (Accardi and Miller, 2004 (link)). Figures were produced in PyMOL Molecular Graphics (DeLano Scientific,
