Hek293s gnti

All constructs were N-terminally tagged with 10xHis followed by EGFP (Ca²⁺ imaging) or mVenus (cryo-EM)^{83 (link)} followed by a human rhinovirus 3 C protease^{84 (link)} cut-site and then human type 3 IP₃R. Plasmids were transformed into DH10Bac cells to generate bacmids^{29 (link)}. 100–200 µg of purified bacmid were incubated with 400 µg of 25,000 MW polyethyleneimine (PEI; Polysciences Cat# 23966) in 1.4 mL water at 55 °C for 30 minutes to sterilize, then added to 50 mL of Sf9 cells at 1×10⁶ cells/mL grown in suspension at 27–30 °C. The Sf9 TNM-FH (Grace’s modified) media was supplemented with 1% penicillin/streptomycin, 0.1% Pluronic F-68 nonionic surfactant (Gibco Cat# 24040), and 4-8% fetal bovine serum to stabilize the virus. Virus titer was amplified to P3 and separated from cell debris by centrifugation. P3 virus was used to infect mammalian HEK293S GnTI^- (ATCC CRL-3022) cells at a density of 3×10⁶ cells/mL at a ratio of 50 mL virus for 800 mL cells and simultaneously stimulated with 4.5 mM valproic acid (VPA; Sigma Cat# P4543). Pellets were harvested from cells by centrifugation at 48–72 hours after infection and snap frozen.

The maltose binding protein fused human PKD2 protein, encompassing residues 53–792 and harboring the F604P GOF mutation^{5 (link)}, was expressed and purified from HEK293S GnTI⁻ (ATCC CRL-3022) cells as previously described^{9 (link)}. In brief, HEK293S GnTI⁻ cells were grown in suspension at 37 °C in freestyle 293 expression medium (Invitrogen, Carlsbad, CA) to the density of ~2 × 10⁶/ml with orbital shaking and then transduced with baculovirus. To boost protein expression, the cell culture was supplemented with sodium butyrate (final concentration of 5 mM) 8–24 h following transduction and maintained at 30 °C. Seveenty-two hours of post-transduction, cells were harvested to obtain crude membrane for affinity purification using amylose resin (New England BioLabs, Ipswich, MA). PKD2 protein was then eluted using buffer containing (in mM) 50 HEPES (pH 7.4), 150 NaCl, 2 TCEP, 0.5 DDM, 20 maltose, and 0.1 mg/ml soybean lipids. PKD2 channels were reconstituted into amphipols A8-35 (Anatrace) as previously described^{37 (link)}. In brief, the purified PKD2 sample was mixed with amphipols A8-35 at 1:3 (w/w) for 4 h and the detergent was removed with Bio-Beads SM-2 which was then removed with a disposable polyprep column. Next, the channel/amphipols mixture was separated with a Superdex 200 column and collected for cryo-EM analysis.