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- PDB-9btu: Human SCNN1B-SCNN1G ENaC dimers -

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Basic information

Entry
Database: PDB / ID: 9btu
TitleHuman SCNN1B-SCNN1G ENaC dimers
Components
  • (10D4 Fab) x 2
  • (Amiloride-sensitive sodium channel subunit ...) x 2
KeywordsMEMBRANE PROTEIN / Ion channel
Function / homology
Function and homology information


sensory perception of salty taste / Sensory perception of salty taste / sensory perception of sour taste / neutrophil-mediated killing of bacterium / aldosterone metabolic process / leukocyte activation involved in inflammatory response / cellular response to vasopressin / sodium channel complex / epithelial fluid transport / mucus secretion ...sensory perception of salty taste / Sensory perception of salty taste / sensory perception of sour taste / neutrophil-mediated killing of bacterium / aldosterone metabolic process / leukocyte activation involved in inflammatory response / cellular response to vasopressin / sodium channel complex / epithelial fluid transport / mucus secretion / sodium ion homeostasis / renal system process / artery smooth muscle contraction / neutrophil activation involved in immune response / cellular response to aldosterone / multicellular organismal-level water homeostasis / potassium ion homeostasis / cellular response to acidic pH / intracellular sodium ion homeostasis / sodium ion import across plasma membrane / ligand-gated sodium channel activity / response to food / erythrocyte homeostasis / WW domain binding / monoatomic ion channel activity / sodium ion transmembrane transport / cytoplasmic vesicle membrane / multicellular organism growth / regulation of blood pressure / Stimuli-sensing channels / gene expression / apical plasma membrane / response to xenobiotic stimulus / external side of plasma membrane / extracellular exosome / nucleoplasm / plasma membrane
Similarity search - Function
Epithelial sodium channel, chordates / Epithelial sodium channel, conserved site / Amiloride-sensitive sodium channels signature. / Epithelial sodium channel / Amiloride-sensitive sodium channel
Similarity search - Domain/homology
Epithelial sodium channel subunit beta / Epithelial sodium channel subunit gamma
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsHouser, A. / Baconguis, I.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM138862 United States
Cystic Fibrosis FoundationBACONG22G0 United States
National Science Foundation (NSF, United States)GVPRS0015B4 United States
CitationJournal: Structure / Year: 2025
Title: Structural insights into subunit-dependent functional regulation in epithelial sodium channels.
Authors: Alexandra Houser / Isabelle Baconguis /
Abstract: Epithelial sodium channels (ENaCs) play a crucial role in Na reabsorption in mammals. To date, four subunits have been identified-α, β, γ, and δ-believed to form different heteromeric complexes. ...Epithelial sodium channels (ENaCs) play a crucial role in Na reabsorption in mammals. To date, four subunits have been identified-α, β, γ, and δ-believed to form different heteromeric complexes. Currently, only the structure of the αβγ complex is known. To investigate the formation of channels with different subunit compositions and to determine how each subunit contributes to distinct channel properties, we co-expressed human δ, β, and γ. Using single-particle cryoelectron microscopy, we observed three distinct ENaC complexes. The structures unveil a pattern in which β and γ positions are conserved among the different complexes while the α position in αβγ trimer is occupied by either δ or another β. The δ subunit induces structural rearrangements in the γ subunit, which may contribute to the differences in channel activity between αβγ and δβγ channels. These structural changes provide molecular insights into how ENaC subunit composition modulates channel function.
History
DepositionMay 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Amiloride-sensitive sodium channel subunit beta
D: 10D4 Fab
C: Amiloride-sensitive sodium channel subunit gamma
E: 10D4 Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,49313
Polymers163,2034
Non-polymers3,2909
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, native gel electrophoresis, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Amiloride-sensitive sodium channel subunit ... , 2 types, 2 molecules BC

#1: Protein Amiloride-sensitive sodium channel subunit beta / Beta-NaCH / Epithelial Na(+) channel subunit beta / ENaCB / Nonvoltage-gated sodium channel 1 ...Beta-NaCH / Epithelial Na(+) channel subunit beta / ENaCB / Nonvoltage-gated sodium channel 1 subunit beta / SCNEB


Mass: 72728.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1B / Plasmid: pGEM BACMID / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: P51168
#3: Protein Amiloride-sensitive sodium channel subunit gamma / Epithelial Na(+) channel subunit gamma / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium channel ...Epithelial Na(+) channel subunit gamma / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium channel 1 subunit gamma / SCNEG


Mass: 74352.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1G / Plasmid: pGEM BACMID / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: P51170

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Antibody , 2 types, 2 molecules DE

#2: Antibody 10D4 Fab


Mass: 9890.183 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): human hybridoma / Production host: Mus musculus (house mouse)
#4: Antibody 10D4 Fab


Mass: 6230.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): human hybridoma / Production host: Mus musculus (house mouse)

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Sugars , 3 types, 9 molecules

#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN
Has protein modificationY
Sequence detailsThe authors state that they do not know which region of the 10D4 Fab is the heavy chain or light ...The authors state that they do not know which region of the 10D4 Fab is the heavy chain or light chain. Furthermore, the Fab sequence and the residue numbering is arbitrary.

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Amiloride-sensitive sodium channel subunits BETA, and GAMMA in complex with 10D4 FabCOMPLEXFab fragment from 10D4 IgG#1-#40MULTIPLE SOURCES
2Human beta subunit isoform 1COMPLEX#11RECOMBINANT
3Human gamma subunit isoform 1COMPLEX#31RECOMBINANT
4Fab fragment from 10D4 IgGCOMPLEX#2, #41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
12NO
23NO
34NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Homo sapiens (human)9606HEK293 GNTI-
23Homo sapiens (human)9606HEK293 GNTI-
34Mus musculus (house mouse)10090
Buffer solutionpH: 7.4
Details: Vitrobot blot parameters were set to a wait time of 0s, blot time of 2s, and a blot force of 1
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMpotassium chlorideKCl1
220 mMHEPESC8H18N2O4S1
35 mMcalcium chlorideCaCl21
SpecimenConc.: 2.275 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse sample of beta and gamma ENaC with 10D4 Fab for the beta subunit
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 295.15 K

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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: SPOT SCAN
Electron lensMode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6021

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3.1particle selectionThis is the version at the end of processing
2SerialEMunkimage acquisitionmultishot multihole was used
4cryoSPARC4.3.1CTF correction
7PHENIX1.2model fitting
9ISOLDE1.6model refinement
10PHENIX1.2model refinement
11cryoSPARC4.3.1initial Euler assignment
12cryoSPARC4.3.1final Euler assignment
13cryoSPARC4.3.1classification
14cryoSPARC4.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1121564
Details: From blob picking in cryoSPARC with a box size of 440 with a maximum particle diameter of 250 and a minimum of 150. Minimum separation distance was 0.6 diameters
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 248323 / Algorithm: FOURIER SPACE
Details: Local Resolution with one of the dimers and the Fabs masked out
Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingPDB-ID: 6wth

6wth


Accession code: 6wth / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058018
ELECTRON MICROSCOPYf_angle_d0.7810903
ELECTRON MICROSCOPYf_dihedral_angle_d11.9112793
ELECTRON MICROSCOPYf_chiral_restr0.0461276
ELECTRON MICROSCOPYf_plane_restr0.0051396

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