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- PDB-7yg1: Cryo-EM structure of the C-terminal domain of the human sodium-ch... -

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Basic information

Entry
Database: PDB / ID: 7yg1
TitleCryo-EM structure of the C-terminal domain of the human sodium-chloride cotransporter
ComponentsSolute carrier family 12 member 3
KeywordsMEMBRANE PROTEIN / transporter / cation-chloride cotransporter
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 / chloride ion homeostasis / renal sodium ion absorption / response to salt / potassium ion 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 / chloride ion homeostasis / renal sodium ion absorption / response to salt / potassium ion homeostasis / response to aldosterone / cell volume homeostasis / sodium ion transport / potassium ion import across plasma membrane / monoatomic ion transport / sodium ion transmembrane transport / chloride transmembrane transport / apical plasma membrane / 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 / SLC12A transporter family / Amino acid permease/ SLC12A domain / 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: 3.77 Å
AuthorsNan, J. / Yang, X.M. / Shan, Z.Y. / Yuan, Y.F. / Zhang, Y.Q.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM structure of the human sodium-chloride cotransporter NCC.
Authors: Jing Nan / Yafei Yuan / Xuemei Yang / Ziyang Shan / Huihui Liu / Feiwen Wei / Wei Zhang / Yanqing Zhang /
Abstract: The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure ...The sodium-chloride cotransporter NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. Here, we present the full-length structure of human NCC, with 2.9 Å for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain. NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in our structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules are indicated to participate in the coordination of ions and TM coupling. Together with transport activity assays, our structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on NCC.
History
DepositionJul 9, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Solute carrier family 12 member 3
B: Solute carrier family 12 member 3


Theoretical massNumber of molelcules
Total (without water)233,8432
Polymers233,8432
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Solute carrier family 12 member 3 / Na-Cl cotransporter / cation-chloride cotransporter


Mass: 116921.461 Da / Num. of mol.: 2 / Mutation: A264G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P55017

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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: Sodium-chloride cotransporter / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8
SpecimenConc.: 9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79225 / Symmetry type: POINT

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