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- EMDB-20537: cryo-EM structure of human NKCC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-20537
Titlecryo-EM structure of human NKCC1
Map dataFinal map, sharpened
Sample
  • Cell: human NKCC1Na-K-Cl cotransporter
    • Protein or peptide: Solute carrier family 12 member 2
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / metal ion transmembrane transporter activity ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / metal ion transmembrane transporter activity / transepithelial chloride transport / potassium ion transmembrane transporter activity / Cation-coupled Chloride cotransporters / ammonium transmembrane transport / sodium ion homeostasis / intracellular chloride ion homeostasis / negative regulation of vascular wound healing / chloride ion homeostasis / cellular response to potassium ion / ammonium transmembrane transporter activity / intracellular potassium ion homeostasis / cell projection membrane / sodium ion import across plasma membrane / intracellular sodium ion homeostasis / potassium ion homeostasis / cellular response to chemokine / T cell chemotaxis / hyperosmotic response / gamma-aminobutyric acid signaling pathway / cell volume homeostasis / regulation of spontaneous synaptic transmission / maintenance of blood-brain barrier / potassium ion import across plasma membrane / transport across blood-brain barrier / sodium ion transmembrane transport / lateral plasma membrane / monoatomic ion transport / chloride transmembrane transport / basal plasma membrane / cell projection / cell periphery / Hsp90 protein binding / cytoplasmic vesicle membrane / extracellular vesicle / cell body / protein-folding chaperone binding / basolateral plasma membrane / neuron projection / apical plasma membrane / neuronal cell body / protein kinase binding / extracellular exosome / membrane / plasma membrane
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
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsCao E / Wang Q / Yang X
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the human cation-chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy.
Authors: Xiaoyong Yang / Qinzhe Wang / Erhu Cao /
Abstract: The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na and/or K gradients to move Cl into or out of cells. NKCC1 is an intensively studied member of the CCC family and ...The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na and/or K gradients to move Cl into or out of cells. NKCC1 is an intensively studied member of the CCC family and plays fundamental roles in regulating trans-epithelial ion movement, cell volume, chloride homeostasis and neuronal excitability. Here, we report a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten transmembrane (TM) helices harboring the transport core and TM11-TM12 helices lining the dimer interface. TM1 and TM6 helices break α-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exits, raising the possibility that K, Na, and Cl ions may traverse along their own routes for translocation. NKCC1 structure provides a blueprint for further probing structure-function relationships of NKCC1 and other CCCs.
History
DepositionAug 1, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseMar 25, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pzt
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

emd_20537.png

Downloads & links

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Map

FileDownload / File: emd_20537.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map, sharpened
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.6
Minimum - Maximum-1.9894207 - 2.4757376
Average (Standard dev.)-0.0010888699 (±0.058002725)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 315.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z315.000315.000315.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-1.9892.476-0.001

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Supplemental data

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Additional map: Final map, unsharpened

Fileemd_20537_additional.map
AnnotationFinal map, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from cryosparc v2

Fileemd_20537_half_map_1.map
Annotationhalf map from cryosparc v2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Another half map from cryosparc v2

Fileemd_20537_half_map_2.map
AnnotationAnother half map from cryosparc v2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human NKCC1

EntireName: human NKCC1Na-K-Cl cotransporter
Components
  • Cell: human NKCC1Na-K-Cl cotransporter
    • Protein or peptide: Solute carrier family 12 member 2

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Supramolecule #1: human NKCC1

SupramoleculeName: human NKCC1 / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Solute carrier family 12 member 2

MacromoleculeName: Solute carrier family 12 member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 131.668484 KDa
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
SequenceString: MEPRPTAPSS GAPGLAGVGE TPSAAALAAA RVELPGTAVP SVPEDAAPAS RDGGGVRDEG PAAAGDGLGR PLGPTPSQSR FQVDLVSEN AGRAAAAAAA AAAAAAAAGA GAGAKQTPAD GEASGESEPA KGSEEAKGRF RVNFVDPAAS SSAEDSLSDA A GVGVDGPN ...String:
MEPRPTAPSS GAPGLAGVGE TPSAAALAAA RVELPGTAVP SVPEDAAPAS RDGGGVRDEG PAAAGDGLGR PLGPTPSQSR FQVDLVSEN AGRAAAAAAA AAAAAAAAGA GAGAKQTPAD GEASGESEPA KGSEEAKGRF RVNFVDPAAS SSAEDSLSDA A GVGVDGPN VSFQNGGDTV LSEGSSLHSG GGGGSGHHQH YYYDTHTNTY YLRTFGHNTM DAVPRIDHYR HTAAQLGEKL LR PSLAELH DELEKEPFED GFANGEESTP TRDAVVTYTA ESKGVVKFGW INGVLVRCML NIWGVMLFIR LSWIVGQAGI GLS VLVIMM ATVVTTITGL STSAIATNGF VRGGRAYYLI SRSLGPEFGG AIGLIFAFAN AVAVAMYVVG FAETVVELLK EHSI LMIDE INDIRIIGAI TVVILLGISV AGMEWEAKAQ IVLLVILLLA IGDFVIGTFI PLESKKPKGF FGYKSEIFNE NFGPD FREE ETFFSVFAIF FPAATGILAG ANISGDLADP QSAIPKGTLL AILITTLVYV GIAVSVGSCV VRDATGNVND TIVTEL TNC TSAACKLNFD FSSCESSPCS YGLMNNFQVM SMVSGFTPLI SAGIFSATLS SALASLVSAP KIFQALCKDN IYPAFQM FA KGYGKNNEPL RGYILTFLIA LGFILIAELN VIAPIISNFF LASYALINFS VFHASLAKSP GWRPAFKYYN MWISLLGA I LCCIVMFVIN WWAALLTYVI VLGLYIYVTY KKPDVNWGSS TQALTYLNAL QHSIRLSGVE DHVKNFRPQC LVMTGAPNS RPALLHLVHD FTKNVGLMIC GHVHMGPRRQ AMKEMSIDQA KYQRWLIKNK MKAFYAPVHA DDLREGAQYL MQAAGLGRMK PNTLVLGFK KDWLQADMRD VDMYINLFHD AFDIQYGVVV IRLKEGLDIS HLQGQEELLS SQEKSPGTKD VVVSVEYSKK S DLDTSKPL SEKPITHKVE EEDGKTATQP LLKKESKGPI VPLNVADQKL LEASTQFQKK QGKNTIDVWW LFDDGGLTLL IP YLLTTKK KWKDCKIRVF IGGKINRIDH DRRAMATLLS KFRIDFSDIM VLGDINTKPK KENIIAFEEI IEPYRLHEDD KEQ DIADKM KEDEPWRITD NELELYKTKT YRQIRLNELL KEHSSTANII VMSLPVARKG AVSSALYMAW LEALSKDLPP ILLV RGNHQ SVLTFYS

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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

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 10.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 90803

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6pzt:
cryo-EM structure of human NKCC1

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