EM Data Acquisition and Processing.

Immediately following size-exclusion chromatography, the CFTR (E1371Q) sample was concentrated to 5 mg/mL (32 µM) and incubated with 8 mM ATP, 10 mM MgCl₂, and 100 µM CFTR_inh-172 on ice for 30 min. Three mM fluorinated Fos-choline-8 was added to the samples directly before application onto glow-discharged Quantifoil R0.6/1 300 mesh Cu grids. Samples were then vitrified using a Vitrobot Mark IV (Field Electron and Ion Company, FEI).

Cryo-EM images were collected in super-resolution mode on a 300 kV Titan Krios (FEI) equipped with a K3 Summit detector (Gatan) using SerialEM (SI Appendix, Table S1). Images were corrected for gain reference and binned by 2. Drift correction was performed using MotionCor2 (57). Contrast transfer function (CTF) estimation was performed using GCTF (58). GCTF values were used for further processing steps. Particles were picked using Gautomatch (https://www.mrc-lmb.cam.ac.uk/kzhang/). All subsequent steps of map reconstruction and resolution estimation were carried out using RELION 3.1 (59) (SI Appendix, Fig. S1).

Following the initial round of 3-dimensional classification, the best class was refined and further classified into four classes and processed until no further improvement was observed (SI Appendix, Fig. S1A). Despite having slightly different nominal resolutions, the top three classes essentially represent the same conformation of CFTR (SI Appendix, Fig. S1B), with the density for the inhibitor best defined in the highest resolution map (compare SI Appendix, Fig. S1B with Fig. 2B).