Supplementary Figure 3: Additional molecular dynamics analysis.

A: Representative view of lipid interaction with a single subunit of the closed MscS structure. Full heptameric structure of closed MscS is shown. A section of the TM domain and the lipids within the pocket is enlarged to show lipid interactions with the polypeptide chain of a single monomer (salmon). The TM3ab helices of the adjacent subunit are shown in yellow, with the hydrophobic gate residues in orange (in space-filling representation). Phospholipids are shown as space-filling representations, with the colours set to distinguish the acyl tails (turquoise) from the headgroups. Note the orientation of the lipids: headgroups pointing downwards with respect to the tails signify a lipid in the cytosolic bilayer leaflet. The opposite orientation signifies a lipid in the periplasmic leaflet (c.f. Fig 3. A, B).

B: Representative view of lipid interaction with a single subunit of the open MscS structure. The heptameric structure of open MscS is shown, with a section of the TM domain and the lipids within the pocket enlarged to show lipid interactions with the polypeptide chain of a single monomer (salmon). The hydrophobic gate residues are shown in orange space-filling representations. Phospholipid representations and significance of orientation as in Fig S3A.

C: Protein proximity to lipid during AT-MD is correlated with proximity to proposed lipid in the crystal structure. Average separation between a single subunit from the new open structure and persistent lipid in the lower region of the pocket during 50-100 ns AT-MD (orange, error bars show one standard deviation) compared to the separation between the new crystal structure and electron density, modelled as carbon (blue).

D: Lipid contacts to the closed and open structures differ significantly. Data were collected from 50 to 100 ns during each AT-MD simulation, and averaged across 5 simulations per state. Statistically different numbers of lipids within 6 Å between closed and open states are shown per residue as heat-mapped residues on the polypeptide chain of the TM domain from a single subunit in the closed structure (p < 0.01). A dark blue residue indicates more contacts to the closed state (maximum 1.7 lipids more) whereas red indicates that the open state has more lipid contacts to the residue (maximum 1.4 lipids more). A white residue indicates no significant difference in contact number between the two states.

E: ‘Persistent’ (those that remain with 6 Å contact of residues in the pockets during the length simulation) and total (including those that are exchanged with the bilayer during the simulation). Dark bars are those in the closed, white in the open structure.

F: (i) The headgroups of the persisting POPE lipids shown in (iii) were analysed for contact preferences to MscS residues. Headgroups make most contacts to charges along the TM1/TM2 helices. ‘Persistent’ lipid configurations in the lower pocket region in the closed (ii) and open (iii) state of MscS. Lipids that make contact (6 Å cutoff) to the lower pocket region throughout 50 to 100 ns AT-MD are displayed in snapshots from the end of the respective simulations. Lipid molecules in the open structure reach the hydrophobic seal (iii), whereas the lipids in the closed structure are prevented from reaching the seal (ii).