Nevertheless, to prove that multiple CNGB1 subunits are coassembled with CNGA1-∆CLZ subunits, we examined the FRET between CNGA and CNGB subunits. For acceptor sensitization, the apparent FRET efficiency increases as the donor-to-acceptor ratio increases 27 . Therefore, if the donor fluorophore (eCFP) was fused to the CNGB subunit and the acceptor fluorophore (eYFP) was fused to the CNGA subunit, then an increase in apparent FRET should be seen if multiple CNGB1 subunits assembled in the channel complex ( Supplementary Fig. S2) . Indeed, the results show a significant increase in FRET between eCFP-CNGB1-∆N and eYFP-CNGA1-∆CLZ compared with eYFP-CNGA1 (Fig. 4e) . These results indicate that multiple CNGB1 subunits are co-assembling with CNGA1-∆CLZ subunits. Overall, it is clear that deletion of the CLZ domain relaxes the 3:1 constraint on subunit assembly.