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- EMDB-40457: Cryo-EM structure of human NCX1 in apo inactivated state -

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Basic information

Entry
Database: EMDB / ID: EMD-40457
TitleCryo-EM structure of human NCX1 in apo inactivated state
Map data
Sample
  • Complex: human cardiac NCX1 in complex with antibody Fab fragment
    • Complex: human cardiac NCX1 in apo inactivated state
      • Protein or peptide: Sodium/calcium exchanger 1
    • Complex: antibody Fab fragment
      • Protein or peptide: Fab light chainFragment antigen-binding
      • Protein or peptide: Fab heavy chainFragment antigen-binding
  • Ligand: CALCIUM IONCalcium
  • Ligand: SODIUM IONSodium
  • Ligand: water
KeywordsNa/Ca exchanger / sodium calcium exchanger / TRANSPORT PROTEIN
Function / homology
Function and homology information


relaxation of smooth muscle / vascular associated smooth muscle contraction / calcium ion export / regulation of cell communication by electrical coupling / calcium:sodium antiporter activity / sodium ion export across plasma membrane / membrane depolarization during cardiac muscle cell action potential / regulation of the force of heart contraction / sodium ion import across plasma membrane / intracellular sodium ion homeostasis ...relaxation of smooth muscle / vascular associated smooth muscle contraction / calcium ion export / regulation of cell communication by electrical coupling / calcium:sodium antiporter activity / sodium ion export across plasma membrane / membrane depolarization during cardiac muscle cell action potential / regulation of the force of heart contraction / sodium ion import across plasma membrane / intracellular sodium ion homeostasis / negative regulation of protein serine/threonine kinase activity / calcium ion import / regulation of cardiac muscle contraction by calcium ion signaling / cardiac muscle cell development / calcium ion transmembrane import into cytosol / calcium ion transport into cytosol / Sodium/Calcium exchangers / cell communication by electrical coupling involved in cardiac conduction / Reduction of cytosolic Ca++ levels / relaxation of cardiac muscle / ankyrin binding / negative regulation of cytosolic calcium ion concentration / cellular response to caffeine / calcium ion import across plasma membrane / positive regulation of the force of heart contraction / regulation of cardiac conduction / sodium ion transmembrane transport / intercalated disc / calcium ion homeostasis / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / positive regulation of bone mineralization / axon terminus / monoatomic ion transport / Ion homeostasis / cardiac muscle contraction / T-tubule / response to muscle stretch / cytoskeletal protein binding / regulation of heart rate / muscle contraction / cell periphery / calcium ion transmembrane transport / sarcolemma / intracellular calcium ion homeostasis / Z disc / cellular response to reactive oxygen species / postsynapse / regulation of gene expression / transmembrane transporter binding / postsynaptic density / calmodulin binding / axon / neuronal cell body / dendrite / synapse / calcium ion binding / nucleoplasm / plasma membrane
Similarity search - Function
Sodium/calcium exchanger, isoform 1 / Sodium/calcium exchanger protein / Sodium/calcium exchanger domain, C-terminal extension / C-terminal extension of sodium/calcium exchanger domain / Na-Ca exchanger/integrin-beta4 / Calx-beta domain / Domains in Na-Ca exchangers and integrin-beta4 / NCX, central ion-binding domain superfamily / Sodium/calcium exchanger membrane region / Sodium/calcium exchanger protein ...Sodium/calcium exchanger, isoform 1 / Sodium/calcium exchanger protein / Sodium/calcium exchanger domain, C-terminal extension / C-terminal extension of sodium/calcium exchanger domain / Na-Ca exchanger/integrin-beta4 / Calx-beta domain / Domains in Na-Ca exchangers and integrin-beta4 / NCX, central ion-binding domain superfamily / Sodium/calcium exchanger membrane region / Sodium/calcium exchanger protein / CalX-like domain superfamily / DnaJ domain
Similarity search - Domain/homology
Sodium/calcium exchanger 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXue J / Jiang Y
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM140892 United States
Welch FoundationI-1578 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural mechanisms of the human cardiac sodium-calcium exchanger NCX1.
Authors: Jing Xue / Weizhong Zeng / Yan Han / Scott John / Michela Ottolia / Youxing Jiang /
Abstract: Na/Ca exchangers (NCX) transport Ca in or out of cells in exchange for Na. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca homeostasis. Although extensively ...Na/Ca exchangers (NCX) transport Ca in or out of cells in exchange for Na. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na and Ca. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca and Na. We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca], a TM-associated four-stranded β-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca binding to the cytosolic second Ca-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins.
History
DepositionApr 12, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40457.png

Downloads & links

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Map

FileDownload / File: emd_40457.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-1.8457354 - 3.1527026
Average (Standard dev.)0.0022356494 (±0.062877364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: focused CBD map of human cardiac NCX1 in apo inactivated state

Fileemd_40457_additional_1.map
Annotationfocused CBD map of human cardiac NCX1 in apo inactivated state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: focused TM map of human cardiac NCX1 in apo inactivated state

Fileemd_40457_additional_2.map
Annotationfocused TM map of human cardiac NCX1 in apo inactivated state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40457_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40457_half_map_2.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 : human cardiac NCX1 in complex with antibody Fab fragment

EntireName: human cardiac NCX1 in complex with antibody Fab fragment
Components
  • Complex: human cardiac NCX1 in complex with antibody Fab fragment
    • Complex: human cardiac NCX1 in apo inactivated state
      • Protein or peptide: Sodium/calcium exchanger 1
    • Complex: antibody Fab fragment
      • Protein or peptide: Fab light chainFragment antigen-binding
      • Protein or peptide: Fab heavy chainFragment antigen-binding
  • Ligand: CALCIUM IONCalcium
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: human cardiac NCX1 in complex with antibody Fab fragment

SupramoleculeName: human cardiac NCX1 in complex with antibody Fab fragment
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: human cardiac NCX1 in apo inactivated state

SupramoleculeName: human cardiac NCX1 in apo inactivated state / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: antibody Fab fragment

SupramoleculeName: antibody Fab fragment / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Sodium/calcium exchanger 1

MacromoleculeName: Sodium/calcium exchanger 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 109.18107 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYNMRRLSLS PTFSMGFHLL VTVSLLFSHV DHVIAETEME GEGNETGECT GSYYCKKGVI LPIWEPQDPS FGDKIARATV YFVAMVYMF LGVSIIADRF MSSIEVITSQ EKEITIKKPN GETTKTTVRI WNETVSNLTL MALGSSAPEI LLSVIEVCGH N FTAGDLGP ...String:
MYNMRRLSLS PTFSMGFHLL VTVSLLFSHV DHVIAETEME GEGNETGECT GSYYCKKGVI LPIWEPQDPS FGDKIARATV YFVAMVYMF LGVSIIADRF MSSIEVITSQ EKEITIKKPN GETTKTTVRI WNETVSNLTL MALGSSAPEI LLSVIEVCGH N FTAGDLGP STIVGSAAFN MFIIIALCVY VVPDGETRKI KHLRVFFVTA AWSIFAYTWL YIILSVISPG VVEVWEGLLT FF FFPICVV FAWVADRRLL FYKYVYKRYR AGKQRGMIIE HEGDRPSSKT EIEMDGKVVN SHVENFLDGA LVLEVDERDQ DDE EARREM ARILKELKQK HPDKEIEQLI ELANYQVLSQ QQKSRAFYRI QATRLMTGAT ENDPVSKIFF EQGTYQCLEN CGTV ALTII RRGGDLTNTV FVDFRTEDGT ANAGSDYEFT EGTVVFKPGD TQKEIRVGII DDDIFEEDEN FLVHLSNVKV SSEAS EDGI LEANHVSTLA CLGSPSTATV TIFDDDHAGI FTFEEPVTHV SESIGIMEVK VLRTSGARGN VIVPYKTIEG TARGGG EDF EDTCGELEFQ NDEIVKTISV KVIDDEEYEK NKTFFLEIGE PRLVEMSEKK ALLLNELGGF TITGKYLFGQ PVFRKVH AR EHPILSTVIT IADEYDDKQP LTSKEEEERR IAEMGRPILG EHTKLEVIIE ESYEFKSTVD KLIKKTNLAL VVGTNSWR E QFIEAITVSA GEDDDDDECG EEKLPSCFDY VMHFLTVFWK VLFAFVPPTE YWNGWACFIV SILMIGLLTA FIGDLASHF GCTIGLKDSV TAVVFVALGT SVPDTFASKV AATQDQYADA SIGNVTGSNA VNVFLGIGVA WSIAAIYHAA NGEQFKVSPG TLAFSVTLF TIFAFINVGV LLYRRRPEIG GELGGPRTAK LLTSCLFVLL WLLYIFFSSL EAYCHIKGFA AAGGSWSHPQ F EKGGGSGG GSGGSAWSHP QFEK

UniProtKB: Sodium/calcium exchanger 1

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Macromolecule #2: Fab light chain

MacromoleculeName: Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 21.740082 KDa
SequenceString: QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNNRVPGV PARFSGSLIG DKAALTITGA QTEDEAIYF CALWYSNHWV FGGGTKLTVL GQPKSSPSVT LFPPSSEELE TNKATLVCTI TDFYPGVVTV DWKVDGTPVT Q GMETTQPS ...String:
QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNNRVPGV PARFSGSLIG DKAALTITGA QTEDEAIYF CALWYSNHWV FGGGTKLTVL GQPKSSPSVT LFPPSSEELE TNKATLVCTI TDFYPGVVTV DWKVDGTPVT Q GMETTQPS KQSNNKYMAS SYLTLTARAW ERHSSYSCQV THEG

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Macromolecule #3: Fab heavy chain

MacromoleculeName: Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.435598 KDa
SequenceString: QVQLQQSGAE LARPGASVKL SCKATGYSFT SYWMQWVKQR PGQGMEWIGA IYPGDVTSRY TQKFKGKATL TADKSSSTAF MQLRSLASE DSAVYYCARW SGYYGSSSFD YWGQGTTLTV SSAKTTPPSV YPLAPGCGDT TGSSVTLGCL VKGYFPESVT V TWNSGSLS ...String:
QVQLQQSGAE LARPGASVKL SCKATGYSFT SYWMQWVKQR PGQGMEWIGA IYPGDVTSRY TQKFKGKATL TADKSSSTAF MQLRSLASE DSAVYYCARW SGYYGSSSFD YWGQGTTLTV SSAKTTPPSV YPLAPGCGDT TGSSVTLGCL VKGYFPESVT V TWNSGSLS SSVHTFPALL QSGLYTMSSS VTVPSSTWPS QTVTCSVAHP ASSTTVDKKL EPSGPISTIN PCPPCKECHK CP APNLEGG PS

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 5 / Number of copies: 3
Molecular weightTheoretical: 22.99 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 133999
FSC plot (resolution estimation)

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