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- EMDB-13008: Mechanosensitive channel MscS solubilized with LMNG in open confo... -

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Basic information

Entry
Database: EMDB / ID: EMD-13008
TitleMechanosensitive channel MscS solubilized with LMNG in open conformation with added lipid
Map datapost-processed: unmasked, sharpened
Sample
  • Complex: homoheptameric complex of MscS coordinated with lipids and detergents DDM and LMNG
    • Protein or peptide: Mechanosensitive channel of small conductance (MscS)
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: DODECYL-BETA-D-MALTOSIDE
Keywordsmechanosensitive channel / LMNG / delipidation / MEMBRANE PROTEIN
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 SE11 (bacteria) / Escherichia coli SE11
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsRasmussen T / Flegler VJ
Funding support Germany, 2 items
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 27, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ooa
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13008.png

Downloads & links

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Map

FileDownload / File: emd_13008.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpost-processed: unmasked, sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 319.05 Å		1.06 Å/pix.
x 300 pix.
= 319.05 Å		1.06 Å/pix.
x 300 pix.
= 319.05 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0635 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.06
Minimum - Maximum-0.2138994 - 0.40267548
Average (Standard dev.)-0.000029790404 (±0.012008952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 319.05002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z319.050319.050319.050
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ640640640
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2140.403-0.000

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Supplemental data

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Mask #1

Fileemd_13008_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half-map

Fileemd_13008_half_map_1.map
Annotationunfiltered half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unfiltered half-map

Fileemd_13008_half_map_2.map
Annotationunfiltered half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : homoheptameric complex of MscS coordinated with lipids and deterg...

EntireName: homoheptameric complex of MscS coordinated with lipids and detergents DDM and LMNG
Components
  • Complex: homoheptameric complex of MscS coordinated with lipids and detergents DDM and LMNG
    • Protein or peptide: Mechanosensitive channel of small conductance (MscS)
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: DODECYL-BETA-D-MALTOSIDE

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Supramolecule #1: homoheptameric complex of MscS coordinated with lipids and deterg...

SupramoleculeName: homoheptameric complex of MscS coordinated with lipids and detergents DDM and LMNG
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli SE11 (bacteria)
Molecular weightTheoretical: 224 KDa

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

MacromoleculeName: Mechanosensitive channel of small conductance (MscS) / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli SE11
Molecular weightTheoretical: 31.994039 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEDLNVVDSI NGAGSWLVAN QALLLSYAVN IVAALAIIIV GLIIARMISN AVNRLMISRK IDATVADFLS ALVRYGIIAF TLIAALGRV GVQTASVIAV LGAAGLAVGL ALQGSLSNLA AGVLLVMFRP FRAGEYVDLG GVAGTVLSVQ IFSTTMRTAD G KIIVIPNG ...String:
MEDLNVVDSI NGAGSWLVAN QALLLSYAVN IVAALAIIIV GLIIARMISN AVNRLMISRK IDATVADFLS ALVRYGIIAF TLIAALGRV GVQTASVIAV LGAAGLAVGL ALQGSLSNLA AGVLLVMFRP FRAGEYVDLG GVAGTVLSVQ IFSTTMRTAD G KIIVIPNG KIIAGNIINF SREPVRRNEF IIGVAYDSDI DQVKQILTNI IQSEDRILKD REMTVRLNEL GASSINFVVR VW SNSGDLQ NVYWDVLERI KREFDAAGIS FPYPQMDVNF KRVKEDKAAL EHHHHHH

UniProtKB: Uncharacterized protein

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Macromolecule #2: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 2 / Number of copies: 7 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM

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Macromolecule #3: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 3 / Number of copies: 21 / Formula: AV0
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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Macromolecule #4: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 4 / Number of copies: 7 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
0.02 %C47H88O22LMNG
0.14 mg/ml1,2-di-(9,10-dibromo)stearoyl-sn-glycero-3-phosphoethanolamine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 150 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 7 sec blotting, blot force +25.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 10080 / Average exposure time: 75.0 sec. / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 262797
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6rld


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: WEIGHTED MAP SUM AT ATOM CENTERS
Output model

PDB-7ooa:
Mechanosensitive channel MscS solubilized with LMNG in open conformation with added lipid

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