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- EMDB-26823: EcMscK G924S mutant in a closed conformation -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-26823
TitleEcMscK G924S mutant in a closed conformation
Map dataEcMscK G924S mutant in a closed conformation
Sample
  • Complex: E.coli MscK
    • Protein or peptide: Mechanosensitive channel MscK
Function / homology
Function and homology information


intracellular water homeostasis / mechanosensitive monoatomic ion channel activity / response to potassium ion / 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, conserved site / Uncharacterized protein family UPF0003 signature. / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel ...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, conserved site / Uncharacterized protein family UPF0003 signature. / Mechanosensitive ion channel MscS, C-terminal / Mechanosensitive ion channel MscS / Mechanosensitive ion channel MscS, transmembrane-2 / Mechanosensitive ion channel / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsMount JW / Yuan P
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_26823.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEcMscK G924S mutant in a closed conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 400 pix.
= 376. Å
0.94 Å/pix.
x 400 pix.
= 376. Å
0.94 Å/pix.
x 400 pix.
= 376. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.022764754 - 2.0292616
Average (Standard dev.)0.0016965995 (±0.026420418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 376.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EcMscK G924S mutant in a closed conformation, half map A

Fileemd_26823_half_map_1.map
AnnotationEcMscK G924S mutant in a closed conformation, half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: EcMscK G924S mutant in a closed conformation, half map B

Fileemd_26823_half_map_2.map
AnnotationEcMscK G924S mutant in a closed conformation, half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : E.coli MscK

EntireName: E.coli MscK
Components
  • Complex: E.coli MscK
    • Protein or peptide: Mechanosensitive channel MscK

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Supramolecule #1: E.coli MscK

SupramoleculeName: E.coli MscK / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Homomeric Heptamer
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Inner Membrane
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant plasmid: PV-1

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Macromolecule #1: Mechanosensitive channel MscK

MacromoleculeName: Mechanosensitive channel MscK / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 127.378789 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MTMFQYYKRS RHFVFSAFIA FVFVLLCQNT AFARASSNGD LPTKADLQAQ LDSLNKQKDL SAQDKLVQQD LTDTLATLDK IDRIKEETV QLRQKVAEAP EKMRQATAAL TALSDVDNDE ETRKILSTLS LRQLETRVAQ ALDDLQNAQN DLASYNSQLV S LQTQPERV ...String:
MTMFQYYKRS RHFVFSAFIA FVFVLLCQNT AFARASSNGD LPTKADLQAQ LDSLNKQKDL SAQDKLVQQD LTDTLATLDK IDRIKEETV QLRQKVAEAP EKMRQATAAL TALSDVDNDE ETRKILSTLS LRQLETRVAQ ALDDLQNAQN DLASYNSQLV S LQTQPERV QNAMYNASQQ LQQIRSRLDG TDVGETALRP SQKVLMQAQQ ALLNAEIDQQ RKSLEGNTVL QDTLQKQRDY VT ANSARLE HQLQLLQEAV NSKRLTLTEK TAQEAVSPDE AARIQANPLV KQELEINQQL SQRLITATEN GNQLMQQNIK VKN WLERAL QSERNIKEQI AVLKGSLLLS RILYQQQQTL PSADELENMT NRIADLRLEQ FEVNQQRDAL FQSDAFVNKL EEGH TNEVN SEVHDALLQV VDMRRELLDQ LNKQLGNQLM MAINLQINQQ QLMSVSKNLK SILTQQIFWV NSNRPMDWDW IKAFP QSLK DEFKSMKITV NWQKAWPAVF IAFLAGLPLL LIAGLIHWRL GWLKAYQQKL ASAVGSLRND SQLNTPKAIL IDLIRA LPV CLIILAVGLI LLTMQLNISE LLWSFSKKLA IFWLVFGLCW KVLEKNGVAV RHFGMPEQQT SHWRRQIVRI SLALLPI HF WSVVAELSPL HLMDDVLGQA MIFFNLLLIA FLVWPMCRES WRDKESHTMR LVTITVLSII PIALMVLTAT GYFYTTLR L AGRWIETVYL VIIWNLLYQT VLRGLSVAAR RIAWRRALAR RQNLVKEGAE GAEPPEEPTI ALEQVNQQTL RITMLLMFA LFGVMFWAIW SDLITVFSYL DSITLWHYNG TEAGAAVVKN VTMGSLLFAI IASMVAWALI RNLPGLLEVL VLSRLNMRQG ASYAITTIL NYIIIAVGAM TVFGSLGVSW DKLQWLAAAL SVGLSFGLQE IFGNFVSGLI ILFERPVRIG DTVTIGSFSG T VSKIRIRA TTITDFDRKE VIIPNKAFVT ERLINWSLTD TTTRLVIRLG VAYGSDLEKV RKVLLKAATE HPRVMHEPMP EV FFTAFGA STLDHELRLY VRELRDRSRT VDELNRTIDQ LCRENDINIA FNQLEVHLHN EKGDEVTEVK RDYKGDDPTP AVG

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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

Concentration7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
150.0 mMKClPotassium Chloride
20.0 mMTris-HClTrisTrizma Base
0.02 %GDNGlyco-Diosgenin

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
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe specimen was homogeneous and monodisperse.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 150000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Average electron dose: 46.16 e/Å2

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 134427
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A / Chain - Residue range: 496-1079
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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