Baculovirus system

The cDNA of full-length OSCA1.2 and OSCA2.2 in A. thaliana was subcloned into the pFastBac1 vector (Invitrogen) with a carboxyl-terminal His₁₀ tag. Primers used for the subcloning can be found in Supplementary Table 1. The recombinant AtOSCA1.2/AtOSCA2.2 was expressed using the baculovirus system (Invitrogen). Briefly, bacmids were generated in DH10Bac cells (Invitrogen). The baculoviruses were generated and amplified in Sf-9 insect cells (Invitrogen). Forty-eight hours after viral infection, cells were collected and resuspended in buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl and 1% (w/v) digitonin, then incubated at 4 °C for 2 h. The insoluble fraction was precipitated by ultracentrifugation at 150,000 × g for 30 min The supernatant was incubated with the Ni-NTA resin (Qiagen) at 4 °C for 30 min The resin was then rinsed three times with wash buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 25 mM imidazole and 0.1% digitonin (w/v). The protein was eluted with wash buffer plus 300 mM imidazole. The eluent was concentrated and then subjected to size-exclusion chromatography using a Superose 6 column (GE Healthcare) in buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl and 0.1% digitonin. The peak fractions were pooled together and further concentrated to approximately 5 mg ml⁻¹ for EM analysis.