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- PDB-6npl: Cryo-EM structure of NKCC1 -

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Basic information

Entry
Database: PDB / ID: 6npl
TitleCryo-EM structure of NKCC1
ComponentsSolute carrier family 12 (sodium/potassium/chloride transporter), member 2
KeywordsMEMBRANE PROTEIN
Function / homology
Function and homology information


swim bladder inflation / Cation-coupled Chloride cotransporters / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / sodium ion homeostasis / chloride ion homeostasis / ammonium transmembrane transporter activity / ear development / potassium ion homeostasis / cell volume homeostasis ...swim bladder inflation / Cation-coupled Chloride cotransporters / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / sodium ion homeostasis / chloride ion homeostasis / ammonium transmembrane transporter activity / ear development / potassium ion homeostasis / cell volume homeostasis / inner ear morphogenesis / potassium ion import across plasma membrane / sodium ion transmembrane transport / chloride transmembrane transport / basolateral plasma membrane / apical plasma membrane / identical protein binding
Similarity search - Function
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 family / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
: / Chem-POV / Solute carrier family 12 member 2 / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFeng, L. / Liao, M.F. / Orlando, B. / Zhang, J.R.
CitationJournal: Nature / Year: 2019
Title: Structure and mechanism of the cation-chloride cotransporter NKCC1.
Authors: Thomas A Chew / Benjamin J Orlando / Jinru Zhang / Naomi R Latorraca / Amy Wang / Scott A Hollingsworth / Dong-Hua Chen / Ron O Dror / Maofu Liao / Liang Feng /
Abstract: Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ...Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.
History
DepositionJan 17, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Aug 28, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
B: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,82715
Polymers201,2872
Non-polymers5,54113
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10850 Å2
ΔGint-127 kcal/mol
Surface area74630 Å2

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Components

#1: Protein Solute carrier family 12 (sodium/potassium/chloride transporter), member 2


Mass: 100643.445 Da / Num. of mol.: 2 / Fragment: AA_permease and SLC12 domains, residues 206-1120
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: slc12a2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0G2KGS0, UniProt: A0A0G2KTI4*PLUS
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#3: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#4: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM

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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: full length co-transporter / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 7.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 96 % / Chamber temperature: 293 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 FIELDBright-field microscopy
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63659 / Symmetry type: POINT

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