Right, the low-resolution cryoEM structure of MscS ∆2–27 (‘Cryst’,~20 Å) shows an overall architecture for the nanodisc-embedded channel. The gray rectangle in the background represents the previous consensus membrane location. Center, relative positioning of 2OAU based on a rigid fit of the structure onto MscS-ND EM density. Left, EM density for the protein (red) and the nanodisc (grey) for MscS-ND, the black ovals highlight the fact that the prominent cavities formed between the TM1-TM2 hairpin and TM3 are fully located outside the membrane.
Black dashed lines depict the limits of the lipid bilayer based on the nanodisc EM density. ( B) Comparison between the location of the membrane interface in MscS-ND, the FC14 crystal structure (2OAU) and the ‘Cryst’ deletion construct. The yellow circle represents the putative average size of the E3D1 nanodisc (~130 Å) in relation to the density, which points to a partial averaging of the density likely due to MscS lateral mobility. Density for the putative heptameric histidine tag complex is shown by a dotted arrow. The location of the bilayer in the nanodisc in indicated by dashed yellow likes (approximately 38 Å in diameter). ( A) Close-up of MscS-ND EM density (in Chimera’s ‘solid’ representation). On the left, a cartoon representation of the TM segments in two adjacent subunits, where the top box indicates the location of the diagram on the right. Shown in sticks and balls representation are residues participating in inter-subunit interactions, either polar in nature (E2–Q11) or hydrophobic (van der Waals) packing (L23–L24, Y27–V29). Highly conserved sites are shown in blue, variable sites in red. ( B) Residue conservation and inter-subunit interactions stabilizing the anchor domain.
The putative location of the lipid bilayer is shown as a pair of dashed lines. Colored grey are regions of the channel resolved in the crystal structure (2OAU), regions newly resolved in the MscS-ND structure are shown in cyan. Right, cartoon diagram showing the MscS monomer. The transparent EM density is shown overlapped to the cartoon of the protein. Bound lipids are shown as stick representation. Each subunit is shown in a different color. ( A) Left, the 3.1 Å resolution structure of the nanodisc-reconstituted (E3D1) MscS heptamer, shown in cartoon representation.
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