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- EMDB-0470: Structure of NKCC1 TM domain -

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Basic information

Entry
Database: EMDB / ID: EMD-0470
TitleStructure of NKCC1 TM domain
Map dataTransmembrane domain of membrane protein
Sample
  • Organelle or cellular component: co-transporter transmembrane domain
    • Protein or peptide: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CHLORIDE IONChloride
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
Solute carrier family 12 member 2 / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFeng L / Liao MF / Orlando B / Zhang JR
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, 2019-
Header (metadata) releaseMar 27, 2019-
Map releaseJul 31, 2019-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0655
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0655
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nph
  • Surface level: 0.0655
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0470.png

Downloads & links

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Map

FileDownload / File: emd_0470.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTransmembrane domain of membrane protein
Voxel sizeX=Y=Z: 1.055 Å
Density
Contour LevelBy AUTHOR: 0.0655 / Movie #1: 0.0655
Minimum - Maximum-0.23401584 - 0.41114736
Average (Standard dev.)-0.00003168363 (±0.012680093)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 232.09999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0551.0551.055
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z232.100232.100232.100
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2340.411-0.000

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Supplemental data

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Additional map: Transmembrane domain of membrane protein

Fileemd_0470_additional.map
AnnotationTransmembrane domain of membrane protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : co-transporter transmembrane domain

EntireName: co-transporter transmembrane domain
Components
  • Organelle or cellular component: co-transporter transmembrane domain
    • Protein or peptide: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CHLORIDE IONChloride

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Supramolecule #1: co-transporter transmembrane domain

SupramoleculeName: co-transporter transmembrane domain / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Solute carrier family 12 (sodium/potassium/chloride transporter),...

MacromoleculeName: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 50.618133 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KFGWIKGVLV RCMLNIWGVM LFIRMTWIVG QAGIAYSCII VIMATVVTTI TGCSTSAIAT NGFVRGGGAY YLISRSLGPE FGGSIGLIF AFANAVAVAM YVVGFAETVV ELLMDSGLLM IDQTNDIRVI GTITVILLLG ISVAGMEWEA KAQIFLLVIL I TAIFNYFI ...String:
KFGWIKGVLV RCMLNIWGVM LFIRMTWIVG QAGIAYSCII VIMATVVTTI TGCSTSAIAT NGFVRGGGAY YLISRSLGPE FGGSIGLIF AFANAVAVAM YVVGFAETVV ELLMDSGLLM IDQTNDIRVI GTITVILLLG ISVAGMEWEA KAQIFLLVIL I TAIFNYFI GSFIAVDSKK KFGFFSYDAG ILAENFGPDF RGQTFFSVFS IFFPAATGIL AGANISGDLA DPQMAIPKGT LL AILITGL VYVGVAISAG ACIVRDATGI ESNFTLISNC TDAACKYGYD FSSCRPTVEG EVSSCKFGLH NDFQVMSVVS GFS PLISAG IFSATLSSAL ASLVSAPKVF QALCKDNIYP GIAIFGKGYG KNNEPLRGYF LTFGIALAFI LIAELNVIAP IISN FFLAS YALINFSVFH ASLANSPGWR PSFKYYNMWA SLAGAILCCV VMFIINWWAA LLTNVIVLSL YIYVSYK

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Macromolecule #2: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 2 / Number of copies: 7 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #4: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7.9 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
0.1 MTirs
0.15 MNaclSodium chloride
0.15 MKcl
0.06 %Digitonin
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 293 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 63659
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6nph:
Structure of NKCC1 TM domain

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