Fig. 4: Anionic phospholipids bind tightly and specifically to the pore.

a Projections along z of coarse grained simulation data showing the number density distribution of lipid head groups surrounding the pore. Source data are provided as a Source data file. b The position of protein associated POPC lipids from 10 randomly selected conducting structures from steered MD superimposed on the crystal structure of the KirBac3.1 pore illustrate a binding cleft in the molecular surface connecting cytosol and fenestrations. POPC lipids are depicted as green sticks, whereas the lipidic fragments depicted in orange arise from the crystal structure. c An isosurface of the fenestration-bound lipids from these calculations demonstrates the relationship of lipid to a canonical lipid-binding site; the isosurface was calculated from 292 lipid molecules at a mass density contour level of 0.15. The isosurface is shown as teal mesh, and sphere representations of the His, Arg and Trp are coloured on an otherwise grey molecular surface. d Native mass spectrum of KirBac3.1 protein in 200 mM ammonium acetate (pH = 8.0) and 0.5% C8E4 (2× the critical micelle concentration) reveals tetrameric complexes with up to four N-terminal methionine truncations (Met- 0, 1, 2, 3 and 4), and with one to three phospholipids binding (+765 Da, +1,513 Da and 2270 Da, respectively). e Lipidomics analysis of KirBac3.1 suggests that PG is enriched amongst the co-purified lipids. f Comparison of the total acyl chain length of PG in KirBac3.1-co-purified lipids and total E. coli lipids suggests that KirBac3.1 preferentially associates with long chain lipids. Error bars represent mean ± SD, with n = 3; individual measurements are overlaid as dot plots. Source data are provided as a Source data file.