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- PDB-8sgt: Cryo-EM structure of human NCX1 in Ca2+ bound, activated state (g... -

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Basic information

Entry
Database: PDB / ID: 8sgt
TitleCryo-EM structure of human NCX1 in Ca2+ bound, activated state (group II in the presence of 0.5 mM Ca2+)
Components
  • Fab heavy chain
  • Fab light chain
  • Sodium/calcium exchanger 1
KeywordsTRANSPORT PROTEIN / Na/Ca exchanger / sodium calcium exchanger
Function / homology
Function and homology information


relaxation of smooth muscle / vascular associated smooth muscle contraction / negative regulation of intracellular signal transduction / calcium:sodium antiporter activity / regulation of cell communication by electrical coupling / calcium ion export / membrane depolarization during cardiac muscle cell action potential / regulation of the force of heart contraction / cell communication by electrical coupling involved in cardiac conduction / sodium ion export across plasma membrane ...relaxation of smooth muscle / vascular associated smooth muscle contraction / negative regulation of intracellular signal transduction / calcium:sodium antiporter activity / regulation of cell communication by electrical coupling / calcium ion export / membrane depolarization during cardiac muscle cell action potential / regulation of the force of heart contraction / cell communication by electrical coupling involved in cardiac conduction / sodium ion export across plasma membrane / intracellular sodium ion homeostasis / sodium ion import across plasma membrane / calcium ion import / calcium ion transport into cytosol / relaxation of cardiac muscle / cardiac muscle cell development / regulation of cardiac muscle contraction by calcium ion signaling / Sodium/Calcium exchangers / ankyrin binding / Reduction of cytosolic Ca++ levels / cellular response to caffeine / negative regulation of cytosolic calcium ion concentration / calcium ion transmembrane import into cytosol / positive regulation of the force of heart contraction / calcium ion import across plasma membrane / intercalated disc / regulation of cardiac conduction / positive regulation of bone mineralization / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium ion homeostasis / cytoskeletal protein binding / cardiac muscle contraction / Ion homeostasis / monoatomic ion transport / response to muscle stretch / axon terminus / T-tubule / sodium ion transmembrane transport / muscle contraction / regulation of heart rate / cell periphery / cellular response to reactive oxygen species / sarcolemma / calcium ion transmembrane transport / Z disc / intracellular calcium ion homeostasis / regulation of gene expression / transmembrane transporter binding / calmodulin binding / postsynapse / postsynaptic density / axon / neuronal cell body / dendrite / calcium ion binding / synapse / 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 / CalX-like domain superfamily / Sodium/calcium exchanger membrane region ...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 / CalX-like domain superfamily / Sodium/calcium exchanger membrane region / NCX, central ion-binding domain superfamily / Sodium/calcium exchanger protein / DnaJ domain
Similarity search - Domain/homology
Sodium/calcium exchanger 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsXue, J. / Jiang, Y.
Funding support United States, 3items
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 13, 2023Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Sodium/calcium exchanger 1
L: Fab light chain
H: Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,5979
Polymers157,3573
Non-polymers2406
Water00
1


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

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Components

#1: Protein Sodium/calcium exchanger 1 / Na(+)/Ca(2+)-exchange protein 1 / Solute carrier family 8 member 1


Mass: 109181.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC8A1, CNC, NCX1 / Production host: Homo sapiens (human) / References: UniProt: P32418
#2: Antibody Fab light chain


Mass: 21740.082 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#3: Antibody Fab heavy chain


Mass: 26435.598 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1human NCX1 in complex with antibody Fab fragmentCOMPLEX#1-#30MULTIPLE SOURCES
2human NCX1 in Ca bound, activated stateCOMPLEX#11RECOMBINANT
3antibody Fab fragmentCOMPLEX#2-#31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112396 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057279
ELECTRON MICROSCOPYf_angle_d0.7529891
ELECTRON MICROSCOPYf_dihedral_angle_d13.3742520
ELECTRON MICROSCOPYf_chiral_restr0.051137
ELECTRON MICROSCOPYf_plane_restr0.0071240

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