Preparation of undocked INX-6 hemichannels for cryo-EM using nanodiscs and GraDeR
This protocol is extracted from research article:
Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids
Sci Adv, Feb 12, 2020; DOI: 10.1126/sciadv.aax3157

The INX-6 hemichannels were reconstituted into nanodiscs following the method described previously (38). Briefly, fractions of purified INX-6 containing hemichannels were mixed with POPC (Avanti Polar Lipids) at 10 mg ml−1 and MSP2N2 at 9.5 mg ml−1. The reconstitution was obtained at a molar ratio of INX-6:MSP2N2:POPC = 1:0.75:100 for WT and 1:0.5:30 for INX-6ΔN. Bio-beads SM-2 resin (Bio-Rad) was added to the mixture to initiate the nanodisc reconstitution reaction by removing the detergents. The mixture was incubated overnight at 4°C with constant rotation. The bio-beads were removed, and the mixture was cleared of debris by ultracentrifugation. The supernatant was loaded onto a Superose 6 Increase 10/300 GL column (GE Healthcare) in buffer without detergent [10 mM tris and 150 mM NaCl (pH 7.5)]. Reconstitution was assessed by fluorescence-detection size exclusion chromatography (45), SDS–polyacrylamide gel electrophoresis, and negative-stain EM. Peak fractions corresponding to reconstituted INX-6 hemichannels in lipid nanodiscs were collected and submitted to cryo-EM.

GraDeR (21) was performed according to a previous study (6), with minor modifications. Lauryl maltose neopentyl glycol (Anatrace) was added to the gel filtration fraction of INX-6 at a final concentration of 0.02%. Buffer A [10 mM tris (pH 7.5), 150 mM NaCl, 5% glycerol, and 0.003% lauryl maltose neopentyl glycol] was used for the top layer, and buffer B [10 mM tris (pH 7.5), 150 mM NaCl, and 25% glycerol] was used for the bottom layer. After stacking the buffers, the gradient was generated with Gradient Master 108 (BioComp Instruments). The solution containing the WT INX-6 hemichannels was loaded on the top of the gradient and centrifuged at 35,000 rpm (209,678g) for 18 hours at 4°C with an SW41Ti rotor. The solution was recovered as fractions using a peristaltic pump and analyzed by fluorescence-detection size exclusion chromatography. The INX-6 solutions were dialyzed against a 10 mM tris (pH 7.5), 50 mM NaCl buffer for 1 hour at 4°C and pooled for cryo-EM. The condition of 50 mM NaCl is also important to facilitate undocked hemichannels.

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