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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 IONPotassium
  • Ligand: CHLORIDE IONChloride
Function / homology
Function and homology information


Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium transmembrane transporter activity / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / plasma membrane => GO:0005886 / chloride transmembrane transport ...Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium transmembrane transporter activity / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / plasma membrane => GO:0005886 / chloride transmembrane transport / chemical synaptic transmission / synapse / protein kinase binding / protein-containing complex
Similarity search - Function
K/Cl co-transporter 4 / K/Cl co-transporter / 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 7
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsReid MS / Kern DM / Brohawn SG
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-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing 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
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  • 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
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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
Voxel sizeX=Y=Z: 1.137 Å
Density
Contour LevelBy AUTHOR: 10.3 / Movie #1: 10.3
Minimum - Maximum-27.97654 - 39.75455
Average (Standard dev.)0.0000000000 (±1)
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 IONPotassium
  • Ligand: CHLORIDE IONChloride

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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

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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 mMEDTAEthylenediaminetetraacetic acid
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.11 sec. / Average electron dose: 0.9333 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1826000
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
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

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