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- PDB-8fht: Cryo-EM structure of human NCC -

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Basic information

Entry
Database: PDB / ID: 8fht
TitleCryo-EM structure of human NCC
ComponentsSolute carrier family 12 member 3
KeywordsMEMBRANE PROTEIN / Membrane transporter
Function / homology
Function and homology information


Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / sodium ion homeostasis / renal sodium ion absorption / chloride ion homeostasis / potassium ion homeostasis / cell volume homeostasis / response to aldosterone ...Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / sodium ion homeostasis / renal sodium ion absorption / chloride ion homeostasis / potassium ion homeostasis / cell volume homeostasis / response to aldosterone / sodium ion transport / potassium ion import across plasma membrane / response to dietary excess / sodium ion transmembrane transport / monoatomic ion transport / chloride transmembrane transport / apical plasma membrane / signal transduction / extracellular exosome / ATP binding / membrane / plasma membrane / cytosol
Similarity search - Function
Thiazide-sensitive Na-K-Cl co-transporter / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 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 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
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
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 30, 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

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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 3
B: Solute carrier family 12 member 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)226,6766
Polymers226,5592
Non-polymers1174
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 3 / Na-Cl cotransporter / NCC / Na-Cl symporter / Thiazide-sensitive sodium-chloride cotransporter


Mass: 113279.719 Da / Num. of mol.: 2 / Mutation: T55D, T60D, S73D, K140N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A3, NCC, TSC / Production host: Homo sapiens (human) / References: UniProt: P55017
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / 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 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
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
CTF correctionType: NONE
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 338556 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037170
ELECTRON MICROSCOPYf_angle_d0.4819794
ELECTRON MICROSCOPYf_dihedral_angle_d3.502988
ELECTRON MICROSCOPYf_chiral_restr0.0381168
ELECTRON MICROSCOPYf_plane_restr0.0031204

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