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- EMDB-11804: Structure of Wild-Type Human Potassium Chloride Transporter KCC3 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11804
TitleStructure of Wild-Type Human Potassium Chloride Transporter KCC3 in NaCl (LMNG/CHS)
Map data
Sample
  • Complex: Homodimeric membrane protein complex
    • Protein or peptide: Human potassium chloride transporter KCC3
Function / homology
Function and homology information


Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / potassium ion transmembrane transporter activity / chloride ion homeostasis / cellular hypotonic salinity response / cellular hypotonic response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane ...Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / potassium ion transmembrane transporter activity / chloride ion homeostasis / cellular hypotonic salinity response / cellular hypotonic response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / cellular response to glucose stimulus / basolateral plasma membrane / angiogenesis / chemical synaptic transmission / axon / synapse / protein kinase binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
K/Cl co-transporter / 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 6
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsChi G / Man H / Pike ACW / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R / Moreau C / Snee M ...Chi G / Man H / Pike ACW / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R / Moreau C / Snee M / Abrusci P / Arrowsmith CH / Bountra C / Edwards AM / Marsden BD / Burgess-Brown NA / Duerr KL
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
European Commission United Kingdom
CitationJournal: EMBO J / Year: 2021
Title: Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.
Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong ...Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong Wang / Gavin McKinley / Christophe P Moreau / Kiran D Bountra / Patrizia Abrusci / Shubhashish M M Mukhopadhyay / Alejandra Fernandez-Cid / Samira Slimani / Julie L Lavoie / Nicola A Burgess-Brown / Ben Tehan / Frank DiMaio / Ali Jazayeri / Paul Isenring / Carol V Robinson / Katharina L Dürr /
Abstract: Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions ...Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
History
DepositionSep 28, 2020-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11804.png

Downloads & links

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Map

FileDownload / File: emd_11804.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 341.44 Å		1.07 Å/pix.
x 320 pix.
= 341.44 Å		1.07 Å/pix.
x 320 pix.
= 341.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.275 / Movie #1: 0.3
Minimum - Maximum-0.85022485 - 1.4028597
Average (Standard dev.)-0.0015363058 (±0.03508574)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 341.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0671.0671.067
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z341.440341.440341.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.8501.403-0.002

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Supplemental data

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Mask #1

Fileemd_11804_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11804_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11804_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Homodimeric membrane protein complex

EntireName: Homodimeric membrane protein complex
Components
  • Complex: Homodimeric membrane protein complex
    • Protein or peptide: Human potassium chloride transporter KCC3

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Supramolecule #1: Homodimeric membrane protein complex

SupramoleculeName: Homodimeric membrane protein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Human potassium chloride transporter KCC3

MacromoleculeName: Human potassium chloride transporter KCC3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPHFTVTKVE DPEEGAAASI SQEPSLADIK ARIQDSDEPD LSQNSITGEH SQLLDDGHKK ARNAYLNNS NYEEGDEYFD KNLALFEEEM DTRPKVSSLL NRMANYTNLT QGAKEHEEAE N ITEGKKKP TKTPQMGTFM GVYLPCLQNI FGVILFLRLT WVVGTAGVLQ ...String:
MPHFTVTKVE DPEEGAAASI SQEPSLADIK ARIQDSDEPD LSQNSITGEH SQLLDDGHKK ARNAYLNNS NYEEGDEYFD KNLALFEEEM DTRPKVSSLL NRMANYTNLT QGAKEHEEAE N ITEGKKKP TKTPQMGTFM GVYLPCLQNI FGVILFLRLT WVVGTAGVLQ AFAIVLICCC CT MLTAISM SAIATNGVVP AGGSYFMISR ALGPEFGGAV GLCFYLGTTF AAAMYILGAI EIF LVYIVP RAAIFHSDDA LKESAAMLNN MRVYGTAFLV LMVLVVFIGV RYVNKFASLF LACV IVSIL AIYAGAIKSS FAPPHFPVCM LGNRTLSSRH IDVCSKTKEI NNMTVPSKLW GFFCN SSQF FNATCDEYFV HNNVTSIQGI PGLASGIITE NLWSNYLPKG EIIEKPSAKS SDVLGS LNH EYVLVDITTS FTLLVGIFFP SVTGIMAGSN RSGDLKDAQK SIPIGTILAI LTTSFVY LS NVVLFGACIE GVVLRDKFGD AVKGNLVVGT LSWPSPWVIV IGSFFSTCGA GLQSLTGA P RLLQAIAKDN IIPFLRVFGH SKANGEPTWA LLLTAAIAEL GILIASLDLV APILSMFFL MCYLFVNLAC ALQTLLRTPN WRPRFRYYHW ALSFMGMSIC LALMFISSWY YAIVAMVIAG MIYKYIEYQ GAEKEWGDGI RGLSLSAARF ALLRLEEGPP HTKNWRPQLL VLLKLDEDLH V KHPRLLTF ASQLKAGKGL TIVGSVIVGN FLENYGEALA AEQTIKHLME AEKVKGFCQL VV AAKLREG ISHLIQSCGL GGMKHNTVVM GWPNGWRQSE DARAWKTFIG TVRVTTAAHL ALL VAKNIS FFPSNVEQFS EGNIDVWWIV HDGGMLMLLP FLLKQHKVWR KCSIRIFTVA QLED NSIQM KKDLATFLYH LRIEAEVEVV EMHDSDISAY TYERTLMMEQ RSQMLRHMRL SKTER DREA QLVKDRNSML RLTSIGSDED EETETYQEKV HMTWTKDKYM ASRGQKAKSM EGFQDL LNM RPDQSNVRRM HTAVKLNEVI VNKSHEAKLV LLNMPGPPRN PEGDENYMEF LEVLTEG LE RVLLVRGGGS EVITIYS

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 3155 / Average electron dose: 37.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2)
Details: While the nominal resolution is 4.02 A on the FSC curve, the map is actually closer to 4.5 - 7.0 A on visual examination.
Number images used: 175990
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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