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- EMDB-20807: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in... -

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Basic information

Entry
Database: EMDB / ID: EMD-20807
TitleCryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs
Map dataPotassium-chloride cotransporter KCC4
Sample
  • Cell: KCC4 in lipid nanodiscs
    • Protein or peptide: Solute carrier family 12 member 7
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
KeywordsPotassium chloride cotransporter / SLC12 / TRANSPORT PROTEIN
Function / homology
Function and homology information


Cation-coupled Chloride cotransporters / chloride:monoatomic cation symporter activity / potassium:chloride symporter activity / potassium ion import across plasma membrane / potassium ion transmembrane transport / chloride transmembrane transport / cellular response to glucose stimulus / protein kinase binding / protein-containing complex / metal ion binding / plasma membrane
Similarity search - Function
K/Cl co-transporter / 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 7
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsReid MS / Kern DM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128263 United States
CitationJournal: Elife / Year: 2020
Title: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs.
Authors: Michelle S Reid / David M Kern / Stephen Graf Brohawn /
Abstract: Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, ...Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the K-Cl cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K and Cl ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.
History
DepositionOct 5, 2019-
Header (metadata) releaseJun 10, 2020-
Map releaseJun 10, 2020-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 10.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ukn
  • Surface level: 10.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20807.png

Downloads & links

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Map

FileDownload / File: emd_20807.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPotassium-chloride cotransporter KCC4
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.14 Å/pix.
x 180 pix.
= 204.66 Å		1.14 Å/pix.
x 180 pix.
= 204.66 Å		1.14 Å/pix.
x 180 pix.
= 204.66 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.137 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 10.300000000000001 / Movie #1: 10.3
Minimum - Maximum-27.97654 - 39.754550000000002
Average (Standard dev.)0.00000000002316 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 204.65999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1371.1371.137
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z204.660204.660204.660
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-27.97739.7550.000

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Supplemental data

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Sample components

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Entire : KCC4 in lipid nanodiscs

EntireName: KCC4 in lipid nanodiscs
Components
  • Cell: KCC4 in lipid nanodiscs
    • Protein or peptide: Solute carrier family 12 member 7
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION

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Supramolecule #1: KCC4 in lipid nanodiscs

SupramoleculeName: KCC4 in lipid nanodiscs / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Solute carrier family 12 member 7

MacromoleculeName: Solute carrier family 12 member 7 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 120.610961 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPTNFTVVPV EARADGAGDE AAERTEEPES PESVDQTSPT PGDGNPRENS PFINNVEVER ESYFEGKNMA LFEEEMDSNP MVSSLLNKL ANYTNLSQGV VEHEEDEDSR RREVKAPRMG TFIGVYLPCL QNILGVILFL RLTWIVGAAG VMESFLIVAM C CTCTMLTA ...String:
MPTNFTVVPV EARADGAGDE AAERTEEPES PESVDQTSPT PGDGNPRENS PFINNVEVER ESYFEGKNMA LFEEEMDSNP MVSSLLNKL ANYTNLSQGV VEHEEDEDSR RREVKAPRMG TFIGVYLPCL QNILGVILFL RLTWIVGAAG VMESFLIVAM C CTCTMLTA ISMSAIATNG VVPAGGSYYM ISRSLGPEFG GAVGLCFYLG TTFAGAMYIL GTIEIFLTYI SPSAAIFQAE TA DGEAAAL LNNMRVYGSC ALALMAVVVF VGVKYVNKLA LVFLACVVLS ILAIYAGVIK TAFAPPDIPV CLLGNRTLAN RNF DTCAKM QVVSNGTVTT ALWRLFCNGS SLGATCDEYF AQNNVTEIQG IPGVASGVFL DNLWSTYSDK GAFVEKKGVS SVPV SEESR PGGLPYVLTD IMTYFTMLVG IYFPSVTGIM AGSNRSGDLK DAQKSIPTGT ILAIVTTSFI YLSCIVLFGA CIEGV VLRD KFGEALQGNL VIGMLAWPSP WVIVIGSFFS TCGAGLQSLT GAPRLLQAIA RDGIIPFLQV FGHGKANGEP TWALLL TAL ICETGILIAS LDSVAPILSM FFLMCYMFVN LACAVQTLLR TPNWRPRFKF YHWTLSFLGM SLCLALMFIC SWYYALF AM LIAGCIYKYI EYRGAEKEWG DGIRGLSLNA ARYALLRVEH GPPHTKNWRP QVLVMLNLDS EQCVKHPRLL SFTSQLKA G KGLTIVGSVL EGTYLDKHVE AQRAEENIRS LMSAEKTKGF CQLVVSSNLR DGASHLIQSA GLGGMKHNTV LMAWPEAWK EADNPFSWKN FVDTVRDTTA AHQALLVAKN IDLFPQNQER FSDGNIDVWW IVHDGGMLML LPFLLRQHKV WRKCRMRIFT VAQVDDNSI QMKKDLQMFL YHLRISAEVE VVEMVENDIS AFTYEKTLMM EQRSQMLKQM QLSKNERERE AQLIHDRNTA S HTTATART QAPPTPDKVQ MTWTKEKLIA EKHRNKDTGP SGFKDLFSLK PDQSNVRRMH TAVKLNGVVL NKSQDAQLVL LN MPGPPKS RQGDENYMEF LEVLTEGLNR VLLVRGGGRE VITIYSSNSL EVLFQ

UniProtKB: Solute carrier family 12 member 7

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 1 / 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: 1 / 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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMPotassium ChlorideKCl
1.0 mMEDTA
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3uL, 1 blot force, 4s blot time, 1s wait time.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.11 sec. / Average electron dose: 0.9333 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1826000
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Number images used: 110143
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)

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