Fig. 2: Mechanical and structural features of TMEM16F.

a Superposition of 78 F-D curves from recombinant N-N2B-16F pulled in the absence of Ca²⁺. Only the unfolding of TMEM16F monomers is shown. b Violin plots of the calculated unfolding work for the N-N2B-16F (n = 78; ochre hue), WT-16F (n = 101; gray hue) and purified murine TMEM16F reconstituted into liposomes (PR-16F, n = 68; blue hue). Average work and standard deviations correspond to 16.4 ± 7.5, 18.4 ± 8.4, and 20.4 ± 8.7 aJ for N-N2B-16F, WT-16F, and PR-16F constructs, respectively. A multimodal distribution with two prominent populations referred to as low (LW) and high unfolding work (HW) was observed in all constructs (arrowheads). c Medium-resolution AFM image of reconstituted TMEM16F protein (n = 4 independent AFM experiments). Individual TMEM16F exposing either extracellular or intracellular side are clearly discernible (white and red arrowheads, respectively). d High-resolution images of two representative TMEM16F dimers exposing the intracellular face. Compact (PR-16F(cd), left; n = 5 independent molecules/experiments) and loose (PR16F(ld), right; n = 5 independent molecules/experiments) configurations with the two protomers further apart were observed. Both molecules were imaged within 24 h with the same HS-AFM scanner, and image area dimensions. e Height profile analysis along the white (dp) and blue (cd and ld) dashed lines (see insets). In the loose configuration, a ~ 1 nm membrane depression is often observed in-between the TMEM16F protomers.