A molecular-dynamics method for simulations in the canonical ensemble. Michaud-Agrawal, N. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. Roux, B. The membrane potential and its representation by a constant electric field in computer simulations. Smart, O. HOLE: a program for the analysis of the pore dimensions of ion channel structural models.
Delcour, A. Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. Yang, Z. Iterative stable alignment and clustering of 2D transmission electron microscope images. Structure 20 , — Frank, J. Buchan, D. Nucleic Acids Res. Download references. We thank D. Rees for critical reading of the manuscript, and M. Ebrahim, J. Sotiris and H. You can also search for this author in PubMed Google Scholar. Correspondence to Thomas Walz. Peer review information Nature thanks the anonymous reviewer s for their contribution to the peer review of this work.
Left panels: ribbon representation of the channel with each subunit shown in a different colour. Right panels: a single subunit with domains labelled. Opening of the channel is accompanied by an increase in the tilt of TM1—TM2 and a rotation about the symmetry axis. Some particles are circled. Scale bar, 50 nm. Side length of individual averages, See Methods and Extended Data Table 1 for details.
Subunits are coloured as in Extended Data Fig. TM1—TM2 and TM3b form a hydrophobic pocket that consists of membrane-buried and solvent-exposed regions yellow dashed box. The grey level of the bar above indicates the confidence level of the prediction. Most hydrophobic residues locate to the upper half of the wheel, while most hydrophilic residues locate to its lower half.
Residue Gln21 forms a break between the end of the amphipathic helix and the beginning of TM1. The MscS structure is shown in ribbon representation and is coloured according to hydrophobicity. At a high contouring threshold 0. No such belt is seen at the periplasmic bilayer boundary. Furthermore, the cytoplasmic bilayer boundary remains largely stationary with decreasing threshold levels, whereas the periplasmic bilayer boundary moves further out as more density is included.
The side chains of Arg46 and Arg74 are shown in stick representation. Scale bar, nm. Five to ten images were collected for each condition. The insets show negative-stain EM images of the two peak fractions. Five images were collected for each peak fraction. The trapezoids represent the overall shape of the TMD in the different conformations. The trapezoid shape is defined by the plane connecting Ser58 and Ala28 on TM1. Trapezoids with larger angles would fit better in flatter membranes.
Activation is followed by transition of the channel population towards a closed state and the experiment ends with the rupture of the membrane patch. None of the other maps has a well-defined TMD. Therefore, none of the maps shows MscS in the open conformation seen in the crystal structure of the AlaVal mutant.
None of the maps shows MscS in the open conformation seen in the crystal structure of the AlaVal mutant. The class averages show different thicknesses for the TMD. The side chains of gating residues Leu and Leu are shown as purple sticks. Densities for the pore lipids and the inverted gatekeeper lipids are shown in red and light green, respectively.
The protein residues are coloured by hydrophobicity as in Extended Data Fig. For lipids other than those in the pockets, only the phosphorous atoms are shown as olive-green spheres. The lipids occupying the pocket formed by the orange subunits are shown in cyan stick representation with the phosphorous atoms as olive-green spheres. On average, each pocket holds 3.
Side length of individual averages, 22 nm. The average diameter of the MscS-containing nanodiscs is The ratio of absorbance at nm MscS and at nm Cy5-labelled PC was measured to estimate lipid removal. The five individual measurements are shown as green dots. The columns show the averages and the error bars represent the standard deviations.
Trajectories were divided into blocks of 0. The s. Morphs between MscS in the closed, open, sub-conducting and desensitized states viewed parallel to the membrane side view. Morphs between MscS in the closed, open, sub-conducting and desensitized states viewed perpendicular to the membrane top view. Lipids inside the MscS channel in the open conformation move to the periphery of the channel, where they associate with the transmembrane helices.
PC lipids were placed into the center of the MscS channel in the open conformation as shown at the beginning of the movie. During the equilibration phase of the MD simulations, the lipids moved towards the periphery of the channel and associated with the transmembrane domains, where they remained during the production phase of the simulations.
MscS is shown in white ribbon representation and the lipid acyl chains in cyan spheres representation. For MscS in the closed conformation, lipids diffuse into the solvent-exposed regions of the hydrophobic pockets. MscS in the closed conformation was embedded into a PC lipid bilayer with no lipids occupying the solvent-exposed regions of the hydrophobic pockets between neighboring subunits. During MD simulations, lipids spontaneously diffused into these regions and remained there for the remainder of the simulations.
The protein is shown as transparent white ribbon with two subunits forming a hydrophobic pocket shown in solid white and orange. For the pore lipids inside the center of the MscS channel and the lipids that remain in the leaflets of the PC bilayer during the course of the MD simulation, only the phosphorous atoms are shown as olive-green spheres. For the lipids that move from the cytoplasmic leaflet to the solvent-exposed region of the hydrophobic pocket in between the white and orange subunits, the carbon atoms are shown in blue, oxygen atoms in red, and the phosphorous atoms in tan.
The conformational cycle of MscS, seen parallel to the membrane. In the resting state, MscS shown in gray with two subunits in blue and yellow is closed and its TMD is extended, matching the thickness of a relaxed membrane. Membrane tension indicated by black arrows induces the gatekeeper lipids light green to dissociate and to either flip or diffuse into the periplasmic membrane leaflet. This defined open conformation captured by the X-ray crystal structure appears to exist only in the context of a detergent micelle, whereas the TMD appears to be highly dynamic in the context of a lipid bilayer.
With persisting membrane tension, the first pocket lipids dark green leave the solvent-exposed regions of the hydrophobic pockets between neighboring subunits. This conformation of MscS in a sub-conducting state was artificially captured by reconstituting the channel in an unphysiologically thin membrane. The TMD of the channel in the sub-conducting state would likely be also highly dynamic similar to the TMD of the channel in the open state.
With persisting membrane tension, the remaining pocket lipids will leave the solvent-exposed pockets not shown and MscS will adopt the desensitized state. The very thin TMD in the desensitized state does not match the hydrophobic thickness of the surrounding membrane, and this hydrophobic mismatch provides a driving force for MscS to return to the relaxed closed conformation once membrane tension subsides.
As MscS relaxes into the resting state, it would pick up fresh pore, pocket and gatekeeper lipids, re-sensitizing the channel to membrane tension. The conformational cycle of MscS, seen perpendicular to the membrane from the periplasm. This diameter does allow some ion conduction, requiring pore lipids red to completely close the pore.
Membrane tension indicated by black arrows induces the gatekeeper lipids light green to dissociate and the pore lipids to move to the periphery of the pore. The dynamic nature of the TMD in the open conformation may allow the pore lipids to leave the inside of the channel.
With persisting membrane tension, as pocket lipids dark green leave the solvent-exposed regions of the hydrophobic pockets between neighboring subunits, MscS transitions into a sub-conducting state. With persisting membrane tension, the remaining pocket lipids leave the solvent-exposed pockets not shown and MscS adopts the desensitized state. When membrane tension subsides, the channel returns to the closed state. Reprints and Permissions. Visualization of the mechanosensitive ion channel MscS under membrane tension.
Download citation. Received : 25 February Accepted : 06 January Published : 10 February Issue Date : 18 February Communications Biology Nature Reviews Microbiology By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
Advanced search. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Subjects Cryoelectron microscopy Membrane biophysics. Abstract Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes 1 , 2 , 3.
Access through your institution. Buy or subscribe. Rent or Buy article Get time limited or full article access on ReadCube. Code availability The custom-written SPIDER scripts used to measure the nanodisc size and the Python codes used to analyse molecular dynamics trajectories are available from the corresponding author on request.
References 1. PubMed Article Google Scholar Acknowledgements We thank D. View author publications. Ethics declarations Competing interests The authors declare no competing interests. Additional information Peer review information Nature thanks the anonymous reviewer s for their contribution to the peer review of this work.
Extended data figures and tables. Extended Data Fig. Supplementary information Supplementary Information This file contains further discussion and supportive results for the findings in this study. Reporting Summary. Video 1 Morphs between MscS in the closed, open, sub-conducting and desensitized states viewed parallel to the membrane side view.
Video 2 Morphs between MscS in the closed, open, sub-conducting and desensitized states viewed perpendicular to the membrane top view. Video 3 Lipids inside the MscS channel in the open conformation move to the periphery of the channel, where they associate with the transmembrane helices.
Video 4 For MscS in the closed conformation, lipids diffuse into the solvent-exposed regions of the hydrophobic pockets. Video 5 The conformational cycle of MscS, seen parallel to the membrane. Video 6 The conformational cycle of MscS, seen perpendicular to the membrane from the periplasm. Rights and permissions Reprints and Permissions. About this article. Cite this article Zhang, Y. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines.
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