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- PDB-8fhp: Cryo-EM structure of human NCC (class 3-1) -

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Basic information

Entry
Database: PDB / ID: 8fhp
TitleCryo-EM structure of human NCC (class 3-1)
ComponentsSolute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
KeywordsMEMBRANE PROTEIN / Membrane transporter
Function / homology
Function and homology information


swim bladder inflation / Cation-coupled Chloride cotransporters / Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / ammonium transmembrane transport / sodium ion homeostasis / ammonium channel activity ...swim bladder inflation / Cation-coupled Chloride cotransporters / Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / ammonium transmembrane transport / sodium ion homeostasis / ammonium channel activity / chloride ion homeostasis / renal sodium ion absorption / ear development / response to salt / potassium ion homeostasis / response to aldosterone / cell volume homeostasis / inner ear morphogenesis / sodium ion transport / potassium ion import across plasma membrane / monoatomic ion transport / sodium ion transmembrane transport / chloride transmembrane transport / basolateral plasma membrane / apical plasma membrane / extracellular exosome / ATP binding / metal ion binding / identical protein binding / membrane / plasma membrane / cytosol
Similarity search - Function
Thiazide-sensitive Na-K-Cl co-transporter / Solute carrier family 12 member 1/2 / Solute carrier family 12 member 2 / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter, C-terminal / Solute carrier family 12 / SLC12A transporter family / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
Polythiazide / Solute carrier family 12 member 2 / Solute carrier family 12 member 3
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsZhang, J. / Fan, M. / Feng, L.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2023
Title: Structure and thiazide inhibition mechanism of the human Na-Cl cotransporter.
Authors: Minrui Fan / Jianxiu Zhang / Chien-Ling Lee / Jinru Zhang / Liang Feng /
Abstract: The sodium-chloride cotransporter (NCC) is critical for kidney physiology. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron, and mutations in the NCC cause ...The sodium-chloride cotransporter (NCC) is critical for kidney physiology. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na-Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.
History
DepositionDec 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
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Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 8, 2023Group: Database references / Category: citation
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Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.4May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
B: Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)241,6706
Polymers240,7442
Non-polymers9264
Water00
1


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

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Components

#1: Protein Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera / Na-K-Cl cotransporter 1 / Na-Cl cotransporter / NCC / Na-Cl symporter / Thiazide-sensitive sodium- ...Na-K-Cl cotransporter 1 / Na-Cl cotransporter / NCC / Na-Cl symporter / Thiazide-sensitive sodium-chloride cotransporter


Mass: 120372.164 Da / Num. of mol.: 2 / Mutation: E240A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish), (gene. exp.) Homo sapiens (human)
Gene: slc12a2, nkcc1, SLC12A3, NCC, TSC / Production host: Homo sapiens (human) / References: UniProt: A0A0G2KTI4, UniProt: P55017
#2: Chemical ChemComp-XZF / Polythiazide / (3S)-6-chloro-2-methyl-1,1-dioxo-3-{[(2,2,2-trifluoroethyl)sulfanyl]methyl}-1,2,3,4-tetrahydro-1lambda~6~,2,4-benzothiadiazine-7-sulfonamide


Mass: 439.882 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H13ClF3N3O4S3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / 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

ComponentName: NCC-polythiazide complex / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Danio rerio (zebrafish)7955
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (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: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95312 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0097504
ELECTRON MICROSCOPYf_angle_d0.74610256
ELECTRON MICROSCOPYf_dihedral_angle_d7.2381020
ELECTRON MICROSCOPYf_chiral_restr0.0481192
ELECTRON MICROSCOPYf_plane_restr0.0061246

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