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- PDB-7oo8: Mechanosensitive channel MscS solubilized with LMNG in closed con... -

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Basic information

Entry
Database: PDB / ID: 7oo8
TitleMechanosensitive channel MscS solubilized with LMNG in closed conformation with added lipid
ComponentsMechanosensitive channel of small conductance (MscS)
KeywordsMEMBRANE PROTEIN / mechanosensitive channel / LMNG / delipidation
Function / homology
Function and homology information


membrane => GO:0016020 / transmembrane transport / plasma membrane
Similarity search - Function
Conserved TM helix / Mechanosensitive ion channel, conserved TM helix / Mechanosensitive ion channel MscS, conserved site / Uncharacterized protein family UPF0003 signature. / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel MscS / Mechanosensitive ion channel, beta-domain / Mechanosensitive ion channel MscS, beta-domain superfamily / LSM domain superfamily
Similarity search - Domain/homology
Uncharacterized protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRasmussen, T. / Flegler, V.J. / Boettcher, B.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)Bo1150/15-1 Germany
German Research Foundation (DFG)INST 93/903-1 FUGG Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Mechanosensitive channel gating by delipidation.
Authors: Vanessa Judith Flegler / Akiko Rasmussen / Karina Borbil / Lea Boten / Hsuan-Ai Chen / Hanna Deinlein / Julia Halang / Kristin Hellmanzik / Jessica Löffler / Vanessa Schmidt / Cihan Makbul ...Authors: Vanessa Judith Flegler / Akiko Rasmussen / Karina Borbil / Lea Boten / Hsuan-Ai Chen / Hanna Deinlein / Julia Halang / Kristin Hellmanzik / Jessica Löffler / Vanessa Schmidt / Cihan Makbul / Christian Kraft / Rainer Hedrich / Tim Rasmussen / Bettina Böttcher /
Abstract: The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the ...The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.
History
DepositionMay 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Mechanosensitive channel of small conductance (MscS)
B: Mechanosensitive channel of small conductance (MscS)
C: Mechanosensitive channel of small conductance (MscS)
D: Mechanosensitive channel of small conductance (MscS)
E: Mechanosensitive channel of small conductance (MscS)
F: Mechanosensitive channel of small conductance (MscS)
G: Mechanosensitive channel of small conductance (MscS)


Theoretical massNumber of molelcules
Total (without water)223,9587
Polymers223,9587
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37620 Å2
ΔGint-232 kcal/mol
Surface area86620 Å2
MethodPISA

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Components

#1: Protein
Mechanosensitive channel of small conductance (MscS)


Mass: 31994.039 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: SE11 / Production host: Escherichia coli (E. coli) / References: UniProt: B6I756

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: homoheptameric complex of MscS coordinated with lipids and detergents DDM and LMNG
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.224 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: MJF641 / Plasmid: pTrc99A
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
30.02 %LMNGC47H88O221
40.14 mg/ml1,2-di-(9,10-dibromo)stearoyl-sn-glycero-3-phosphoethanolamine1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 7 sec blotting, blot force +25

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 75 sec. / Electron dose: 80 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3292

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2RELION3.1particle selection
3EPUimage acquisition
5CTFFIND4CTF correction
8Coot0.9.2model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43500 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: WEIGHTED MAP SUM AT ATOM CENTERS
Atomic model buildingPDB-ID: 6RLD

6rld


Accession code: 6RLD / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314098
ELECTRON MICROSCOPYf_angle_d0.37319117
ELECTRON MICROSCOPYf_dihedral_angle_d9.9575110
ELECTRON MICROSCOPYf_chiral_restr0.0392345
ELECTRON MICROSCOPYf_plane_restr0.0022429

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