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- EMDB-26876: Locally refined core of EcMscK G924S in an intermediate conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-26876
TitleLocally refined core of EcMscK G924S in an intermediate conformation
Map dataLocally refined core of EcMscK G924S in an intermediate conformation
Sample
  • Complex: EcMscK in an intermediate conformation
    • Protein or peptide: EcMscK
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 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 8, 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

emd_26876.png

Downloads & links

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Map

FileDownload / File: emd_26876.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally refined core of EcMscK G924S in an intermediate conformation
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.027184045 - 3.033895
Average (Standard dev.)0.0010200812 (±0.01925883)
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: Half A

Fileemd_26876_half_map_1.map
AnnotationHalf A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half B

Fileemd_26876_half_map_2.map
AnnotationHalf B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EcMscK in an intermediate conformation

EntireName: EcMscK in an intermediate conformation
Components
  • Complex: EcMscK in an intermediate conformation
    • Protein or peptide: EcMscK

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Supramolecule #1: EcMscK in an intermediate conformation

SupramoleculeName: EcMscK in an intermediate conformation / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Homomeric Heptamer
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Komagataella pastoris (fungus)

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Macromolecule #1: EcMscK

MacromoleculeName: EcMscK / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
SequenceString: MTMFQYYKRS RHFVFSAFIA FVFVLLCQNT AFARASSNGD LPTKADLQAQ LDSLNKQKDL SAQDKLVQQ DLTDTLATLD KIDRIKEETV QLRQKVAEAP EKMRQATAAL TALSDVDNDE E TRKILSTL SLRQLETRVA QALDDLQNAQ NDLASYNSQL VSLQTQPERV ...String:
MTMFQYYKRS RHFVFSAFIA FVFVLLCQNT AFARASSNGD LPTKADLQAQ LDSLNKQKDL SAQDKLVQQ DLTDTLATLD KIDRIKEETV QLRQKVAEAP EKMRQATAAL TALSDVDNDE E TRKILSTL SLRQLETRVA QALDDLQNAQ NDLASYNSQL VSLQTQPERV QNAMYNASQQ LQ QIRSRLD GTDVGETALR PSQKVLMQAQ QALLNAEIDQ QRKSLEGNTV LQDTLQKQRD YVT ANSARL EHQLQLLQEA VNSKRLTLTE KTAQEAVSPD EAARIQANPL VKQELEINQQ LSQR LITAT ENGNQLMQQN IKVKNWLERA LQSERNIKEQ IAVLKGSLLL SRILYQQQQT LPSAD ELEN MTNRIADLRL EQFEVNQQRD ALFQSDAFVN KLEEGHTNEV NSEVHDALLQ VVDMRR ELL DQLNKQLGNQ LMMAINLQIN QQQLMSVSKN LKSILTQQIF WVNSNRPMDW DWIKAFP QS LKDEFKSMKI TVNWQKAWPA VFIAFLAGLP LLLIAGLIHW RLGWLKAYQQ KLASAVGS L RNDSQLNTPK AILIDLIRAL PVCLIILAVG LILLTMQLNI SELLWSFSKK LAIFWLVFG LCWKVLEKNG VAVRHFGMPE QQTSHWRRQI VRISLALLPI HFWSVVAELS PLHLMDDVLG QAMIFFNLL LIAFLVWPMC RESWRDKESH TMRLVTITVL SIIPIALMVL TATGYFYTTL R LAGRWIET VYLVIIWNLL YQTVLRGLSV AARRIAWRRA LARRQNLVKE GAEGAEPPEE PT IALEQVN QQTLRITMLL MFALFGVMFW AIWSDLITVF SYLDSITLWH YNGTEAGAAV VKN VTMGSL LFAIIASMVA WALIRNLPGL LEVLVLSRLN MRQGASYAIT TILNYIIIAV GAMT VFGSL GVSWDKLQWL AAALSVGLSF GLQEIFGNFV SGLIILFERP VRIGDTVTIG SFSGT VSKI RIRATTITDF DRKEVIIPNK AFVTERLINW SLTDTTTRLV IRLGVAYGSD LEKVRK VLL KAATEHPRVM HEPMPEVFFT AFGASTLDHE LRLYVRELRD RSRTVDELNR TIDQLCR EN DINIAFNQLE VHLHNEKGDE VTEVKRDYKG DDPTPAVG

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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-HClTris
0.02 %Glyco-Diosgenin

Details: Micrographs were pooled from protein purified in the presence of either 150 mM NaCl or 150 mM KCl. 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 sample 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) / Average electron dose: 46.16 e/Å2

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 136195
FSC plot (resolution estimation)

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