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- PDB-9pif: Cryo-EM structure of human NCC with chlorthalidone -

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Basic information

Entry
Database: PDB / ID: 9pif
TitleCryo-EM structure of human NCC with chlorthalidone
Components(Solute carrier family 12 member ...) x 2
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 / sodium ion homeostasis / ammonium transmembrane transport / 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 / sodium ion homeostasis / ammonium transmembrane transport / ammonium channel activity / chloride ion homeostasis / ear development / renal sodium ion absorption / 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 / chloride transmembrane transport / sodium ion transmembrane transport / basolateral plasma membrane / apical plasma membrane / extracellular exosome / ATP binding / membrane / metal ion binding / identical protein binding / 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
: / ADENOSINE-5'-TRIPHOSPHATE / Solute carrier family 12 member 2 / Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Danio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsZhang, J. / Feng, L.
Funding support United States, 3items
OrganizationGrant numberCountry
The G. Harold and Leila Y. Mathers Foundation United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Other private United States
CitationJournal: Nat Commun / Year: 2025
Title: Molecular mechanisms of thiazide-like diuretics-mediated inhibition of the human Na-Cl cotransporter.
Authors: Chien-Ling Lee / Jianxiu Zhang / Liang Feng /
Abstract: Thiazide-type and thiazide-like diuretics are structurally distinct first-line antihypertensive drugs that target the sodium-chloride cotransporter (NCC) in the kidney. Thiazide-like diuretics are ...Thiazide-type and thiazide-like diuretics are structurally distinct first-line antihypertensive drugs that target the sodium-chloride cotransporter (NCC) in the kidney. Thiazide-like diuretics are reported to have better cardioprotective effects than thiazide-type diuretics, but whether this is due to differences in NCC-inhibition mechanisms, if there is any, remains unclear. To understand the molecular mechanisms of NCC inhibition by thiazide-like diuretics, we determine the structures of human NCC (hNCC) bound to two of the most widely used thiazide-like diuretics, chlorthalidone and indapamide, using cryogenic electron microscopy (cryo-EM). Structural analyses reveal shared features and distinctions between NCC-inhibition by thiazide-like and thiazide-type diuretics. Furthermore, structural comparisons allow us to identify polymorphisms in hNCC that have substantial differential effects on the potencies of specific thiazide-like and thiazide-type diuretics. Our work provides important insights into the molecular pharmacology of NCC and a blueprint for developing precision medicine to manage hypertension with thiazide-like and thiazide-type diuretics.
History
DepositionJul 10, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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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
C: Solute carrier family 12 member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)221,1978
Polymers219,2833
Non-polymers1,9135
Water362
1


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

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Components

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Solute carrier family 12 member ... , 2 types, 3 molecules ABC

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


Mass: 98893.148 Da / Num. of mol.: 2 / Mutation: E240A
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: Protein Solute carrier family 12 member 2 / Bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2 / BSC2 / Na-K-Cl cotransporter 1 / NKCC1


Mass: 21497.146 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: slc12a2, nkcc1 / Production host: Homo sapiens (human) / References: UniProt: A0A0G2KTI4

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Sugars , 1 types, 1 molecules

#5: 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.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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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Non-polymers , 3 types, 6 molecules

#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-A1CIB / 2-chloro-5-[(1R)-1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl]benzene-1-sulfonamide / chlorthalidone


Mass: 338.766 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H11ClN2O4S / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

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-chlorthalidone complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 8 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: TFS 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

EM software
IDNameCategory
1Topazparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96877 / Symmetry type: POINT
RefinementHighest resolution: 3.24 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214079
ELECTRON MICROSCOPYf_angle_d0.42619144
ELECTRON MICROSCOPYf_dihedral_angle_d6.8931918
ELECTRON MICROSCOPYf_chiral_restr0.0382170
ELECTRON MICROSCOPYf_plane_restr0.0032378

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