Cholesteryl hemisuccinate tris salt

The human KCNQ1 gene with a C-terminal strep tag was expressed in HEK293S cells at 30 °C for 48 h using the BacMam system. Protein was extracted with 1.5% n-dodecyl-β-D-maltoside (Anatrace) and 0.3% cholesteryl hemisuccinate Tris salt (Anatrace) at 4 °C for 3 h. After purification with Strep-Tactin Sepharose resin (IBA) and size-exclusion chromatography on a Superose 6 10/300 GL column (GE Healthcare), the final sample was concentrated to 8 mg/mL for single-particle analysis. The cryo-EM grids were prepared using a Mark IV Vitrobot (FEI). Micrographs were acquired with a Titan Krios microscope (FEI) operated at 300 kV with a K2 summit direct electron detector (Gatan) via SerialEM (54 (link)) following standard procedure. The defocus range was set from –1.1 to –1.3 μm. Data were dose fractionated to 40 frames with a dose rate of 8 e⁻/pixel/s and a total exposure time of 8 s, corresponding to a total dose of ∼64 e⁻/Ų. Image processing and three-dimensional reconstruction were performed following standard procedures, and the final resolution was estimated by the gold-standard Fourier shell correlation = 0.143 criterion (SI Appendix, Figs. S1–S4 and Table S1). The structure of human KCNQ1-CaM complex (PDB: 6UZZ) (8 (link)) was used as the initial model for model building.