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- EMDB-0473: Cryo-EM structure of NKCC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-0473
TitleCryo-EM structure of NKCC1
Map dataMembrane protein
Sample
  • Organelle or cellular component: full length co-transporter
    • Protein or peptide: Solute carrier family 12 (sodium/potassium/chloride transporter), member 2
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
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 / ammonium transmembrane transport / sodium ion homeostasis / chloride ion homeostasis / ammonium channel activity / ear development / potassium ion homeostasis ...swim bladder inflation / Cation-coupled Chloride cotransporters / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / ammonium transmembrane transport / sodium ion homeostasis / chloride ion homeostasis / ammonium channel activity / ear development / potassium ion homeostasis / cell volume homeostasis / inner ear morphogenesis / potassium ion import across plasma membrane / chloride transmembrane transport / sodium ion transmembrane transport / basolateral plasma membrane / apical plasma membrane / identical protein binding / metal ion 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, 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 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
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-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.056
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.056
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6npl
  • Surface level: 0.056
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0473.png

Downloads & links

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Map

FileDownload / File: emd_0473.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMembrane protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 220 pix.
= 232.1 Å		1.06 Å/pix.
x 220 pix.
= 232.1 Å		1.06 Å/pix.
x 220 pix.
= 232.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.055 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.056 / Movie #1: 0.056
Minimum - Maximum-0.03274331 - 0.32720813
Average (Standard dev.)0.0025884937 (±0.010076064)
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-0.0330.3270.003

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Supplemental data

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Additional map: Membrane protein

Fileemd_0473_additional.map
AnnotationMembrane 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 : full length co-transporter

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

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Supramolecule #1: full length co-transporter

SupramoleculeName: full length co-transporter / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)

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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: 100.643445 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 YIYVSYKKPD VNWGS STQA LTYHQALTHS LQLCGVADHI KTFRPQCLVM TGAPNSRPAI LHLVHAFTKN VGLMLCGHVR ISSRRPNFKE LNSDML RYQ RWLLNNNSKA FYTCVVAEDL RQGTQYMLQA AGLGRLRPNT LVIGFKNDWR IGDIKEVETY INLIHDAFDF QYGVVIL RL REGLDISHIQ GQDDSSGMKD VVVSVDISKD SDGDSSKPSS KATSVQNSPA VQKDKKSPTV PLNVADQRLL DSQQFQQK Q GKGTVDVWWL FDDGGLTLLI PYLIANKKKW KDCKIRVFIG GKINRIDHDR RAMATLLSKF RIDFSDITVL GDINTKPKS EGLTEFAEMI EPYKLREDDM EQEAAEKLKS EEPWRITDNE LELYKAKGNR QIRLNELLKE HSSTANLIVM SMPLARKGAV SSALYMAWL DTLSKDLPPI LLVRGNHQSV LTFYS

UniProtKB: Solute carrier family 12 member 2

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Macromolecule #2: POTASSIUM ION

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

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #4: (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: 4 / 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

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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
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 293 K / Instrument: LEICA EM GP

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Electron microscopy

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

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 63659
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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