Fig. 7

Structure of nhTMEM16 L302A in lipid nanodiscs. a Masked cryo-EM density maps of L302A nhTMEM16 in the presence of 0.5 mM Ca²⁺. One monomer is shown in gray, the other is teal. b Atomic model of L302A nhTMEM16. One monomer is gray, in the other the cytosolic domain is orange, the permeation pathway is green, and the remainder of the protein is blue. Ca²⁺ ions are shown as red spheres. c–f Structural comparison of the lipid pathway of wt (4WIS, left panels, gray) and L302A (right panels, teal) nhTMEM16. The color scheme is the same throughout the figure. c The lipid pathway (TM3-TM6) of the L302A mutant and WT (4WIS) viewed from the plane of the membrane. d–f Close up views of the structural rearrangements induced by the L302A mutation at the lipid pathway. The protein backbone is shown in ribbon representation and important residues are shown as sticks and colored in CPK yellow. d L302A disrupts the TM3/TM4 interface. Dashed lines indicate the distances between L302 on TM3 and I343/L347 on TM4. The distances respectively change from 3.7 to 7.4 Å and from 5 to 7.2 Å indicating that TM4 moves away from TM3. e Closure of the pathway to the membrane. The movement of TM4 towards TM6 enables the packing of the side chains of T333/V337 on TM4 and Y349/T443/V447 on TM6 to form a steric barrier to lipid entry. f Opening of the extracellular gate. The rearrangements induced by the L302A mutation cause the disengagement of E313 and E318 on TM3 from R432 on TM6. The R432-E313 distance increases from 4.9 Å (WT) to 8.9 Å (L302A) while the R432-E318 distance increases from 4.5 Å (WT) to 12.3 Å (L302A). g The diameter of the L302A groove was estimated using the HOLE program⁵⁰. Purple denotes areas of diameter d > 5.5 Å and green areas where 2.75 < d < 5.5 Å. h Top view of the lipid pathway. TM3–6 are shown as cartoon helices. Gray: Ca²⁺-bound open nhTMEM16 (4WIS); pink: Ca²⁺-bound closed nhTMEM16 (6QMB); yellow: Ca²⁺-bound intermediate (6QMA); teal: L302A nhTMEM16