Fig. 4: Effect of mutations on the structure of YnaI in DDM and on the function of YnaI in soy polar liposomes.

a Cryo-EM analysis of A155V mutant YnaI in DDM revealed two different conformations, which are overlaid and shown parallel to the membrane (complete channels) and perpendicular to the membrane (section). One conformation shows the canonical closed conformation (one subunit shown as green-yellow ribbons and the other six subunits as white ribbons), whereas the other conformation shows a rotation of the peripheral helices (one subunit shown as sea green ribbons), but the channel remains closed. Residue Ala155 that was mutated to Val is represented as red spheres. b The pressure sensitivity of wild-type (WT) YnaI (n = 5), WT MscS (n = 6) and YnaI mutants A155V (n = 4), K108L (n = 6), R120A (n = 4) and F40A (n = 3) relative to WT MscL measured in giant spheroplasts prepared from E. coli strain MJF431 (which does not express MscS, MscK and MscM) shows that the pressure sensitivity of YnaI is similar to that of MscL. Mutations R120A and F40A had no effect on the pressure sensitivity of YnaI, whereas mutation K108L and potentially A155V made the channel slightly more sensitive to pressure. Data are presented as mean ± s.e.m. Statistical significance assessed using unpaired two-sided Student’s t-test. c Interactions of pocket lipids (salmon sticks) with YnaI (cyan ribbons) in DOPC nanodiscs. The YnaI residues interacting with the pocket lipids that were mutated are shown as light-green spheres. The inset shows a magnified view of a single hydrophobic pocket. The side chains of the YnaI residues interacting with the pocket lipids that were mutated are shown as light-green sticks.