A synthetic gene fragment encoding residues 1 to 765 (excluding amino acids 766 to 975) of mouse TRPC5 was cloned into the pEGBacMam vector. The resulting protein contains a maltose-binding protein tag on its N terminus. P3 baculoviruses were produced in the Bac-to-Bac Baculovirus Expression System (Invitrogen). HEK293S GnTI [from the American Type Culture Collection (ATCC)] cells were infected with 10% (v/v) P3 baculovirus at a density of 2.0 to 3.0 × 106 cells/ml for protein expression at 37°C. After 24 hours, 10 mM sodium butyrate was added, and the temperature was reduced to 30°C for 72 hours before harvesting. Cells were gently disrupted and resuspended in a solution containing 30 mM Hepes, 150 mM NaCl, and 1 mM DTT (pH 7.5) with EDTA-free protease inhibitor cocktail (Roche). Cells were solubilized for 2 to 3 hours in a solution containing 1.0% (w/v) N-dodecyl-β-d-maltopyranoside (Affymetrix), 0.1% (w/v) CHS (Sigma-Aldrich), 30 mM Hepes, 150 mM NaCl, and 1 mM DTT (pH 7.5) with EDTA-free protease inhibitor cocktail (Roche). After 30 min, the cell lysate was then centrifuged for 60 min at 100,000g, and the supernatant was incubated in amylose resin (New England Biolabs) at 4°C overnight. The resin was washed with 20 column volumes of wash buffer [25 mM Hepes, 150 mM NaCl, 0.1% (w/v) digitonin, 0.01% (w/v) CHS, and 1 mM DTT (pH 7.5) with EDTA-free protease inhibitor cocktail (Roche)]. The protein was eluted with four column volumes of wash buffer with 40 mM maltose. The protein was then concentrated to 0.5 ml and mixed with PMAL-C8 (Anatrace) at 1:4 (w/w) with gentle agitation for 4 hours. Bio-Beads SM-2 (50 mg/ml, reconstitution mixture; Bio-Rad) were added to remove additional detergent; a disposable Poly-Prep column was used to remove Bio-Beads. After incubation at 4°C overnight, the protein was further purified on a Superose 6 size exclusion column in 25 mM Hepes, 150 mM NaCl, and 1 mM DTT (pH 7.5). All purification procedures were carried out either on ice or at 4°C. The peak fractions corresponding to tetrameric TRPC5 was concentrated to 7.0 mg/ml and used for preparation of cryo-EM sample grids.

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