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- PDB-7uw5: EcMscK G924S mutant in a closed conformation -

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Basic information

Entry
Database: PDB / ID: 7uw5
TitleEcMscK G924S mutant in a closed conformation
ComponentsMechanosensitive channel MscK
KeywordsTRANSPORT PROTEIN / MEMBRANE PROTEIN / MECHANOSENSATION / ION CHANNEL
Function / homology
Function and homology information


intracellular water homeostasis / response to potassium ion / mechanosensitive monoatomic ion channel activity / potassium ion transport / plasma membrane
Similarity search - Function
Mechanosensitive ion channel MscS, porin domain / Mechanosensitive ion channel inner membrane domain 1 / Mechanosensitive ion channel inner membrane domain 1 / Mechanosensitive ion channel porin domain / : / : / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel, transmembrane helices 2/3 / Mechanosensitive ion channel MscS, conserved site / Uncharacterized protein family UPF0003 signature. ...Mechanosensitive ion channel MscS, porin domain / Mechanosensitive ion channel inner membrane domain 1 / Mechanosensitive ion channel inner membrane domain 1 / Mechanosensitive ion channel porin domain / : / : / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel, transmembrane helices 2/3 / 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 / P-type ATPase, transmembrane domain superfamily
Similarity search - Domain/homology
Mechanosensitive channel MscK
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsMount, J.W. / Yuan, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS099341 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for mechanotransduction in a potassium-dependent mechanosensitive ion channel.
Authors: Jonathan Mount / Grigory Maksaev / Brock T Summers / James A J Fitzpatrick / Peng Yuan /
Abstract: Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst ...Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst these channels, MscK is unique in that its activation also requires external potassium ions. To better understand this dual gating mechanism by force and ligand, we elucidate distinct structures of MscK along the gating cycle using cryo-electron microscopy. The heptameric channel comprises three layers: a cytoplasmic domain, a periplasmic gating ring, and a markedly curved transmembrane domain that flattens and expands upon channel opening, which is accompanied by dilation of the periplasmic ring. Furthermore, our results support a potentially unifying mechanotransduction mechanism in ion channels depicted as flattening and expansion of the transmembrane domain.
History
DepositionMay 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mechanosensitive channel MscK
B: Mechanosensitive channel MscK
C: Mechanosensitive channel MscK
D: Mechanosensitive channel MscK
E: Mechanosensitive channel MscK
F: Mechanosensitive channel MscK
G: Mechanosensitive channel MscK


Theoretical massNumber of molelcules
Total (without water)891,6527
Polymers891,6527
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Mechanosensitive channel MscK / Potassium efflux system KefA


Mass: 127378.789 Da / Num. of mol.: 7 / Mutation: G924S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: mscK, aefA, kefA, b0465, JW0454 / Production host: Komagataella pastoris (fungus) / References: UniProt: P77338

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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: E.coli MscK / Type: COMPLEX / Details: Homomeric Heptamer / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Cellular location: Inner Membrane
Source (recombinant)Organism: Komagataella pastoris (fungus) / Plasmid: PV-1
Buffer solutionpH: 8
Details: Micrographs were pooled from protein purified in the presence of either 150mM NaCl or 150mM KCl, respectively. Buffers were prepared fresh, degassed, and filtered through a 0.45 um Durapore PVDF membrane
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaClSodium chloride1
2150 mMPotassium ChlorideKCl1
320 mMTrizma BaseTris-HClTris1
40.02 %Glyco-DiosgeninGDN1
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The specimen was homogeneous and monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 150000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 46.16 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 3.1 / Category: 3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134427 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7UW5

Pdb chain-ID: A / Pdb chain residue range: 496-1079

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