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- EMDB-14709: Cryo-EM structure of human NKCC1 (TM domain) -

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Basic information

Entry
Database: EMDB / ID: EMD-14709
TitleCryo-EM structure of human NKCC1 (TM domain)
Map dataSharpened map of human NKCC1 transmembrane domains
Sample
  • Complex: Dimeric NKCC1 in complex with Na+, K+ and 2Cl-
    • Complex: NKCC1 in complex with Na+, K+ and 2Cl-
      • Protein or peptide: Solute carrier family 12 member 2
    • Complex: NKCC1 in complex with Na+, K+ and 2Cl-
  • Ligand: SODIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / metal ion transmembrane transporter activity / 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 ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / metal ion transmembrane transporter activity / 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 / 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 / cell projection membrane / ammonium channel activity / intracellular potassium ion homeostasis / potassium ion homeostasis / T cell chemotaxis / cellular response to chemokine / sodium ion import across plasma membrane / intracellular sodium ion homeostasis / hyperosmotic response / cell volume homeostasis / gamma-aminobutyric acid signaling pathway / 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 / protein-folding chaperone binding / cell body / 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: 2.55 Å
AuthorsNissen P / Fenton R / Neumann C / Lindtoft Rosenbaek L / Kock Flygaard R / Habeck M / Lykkegaard Karlsen J / Wang Y / Lindorff-Larsen K / Gad H ...Nissen P / Fenton R / Neumann C / Lindtoft Rosenbaek L / Kock Flygaard R / Habeck M / Lykkegaard Karlsen J / Wang Y / Lindorff-Larsen K / Gad H / Hartmann R / Lyons J
Funding support Denmark, France, 6 items
OrganizationGrant numberCountry
Lundbeckfonden2015-3225 Denmark
LundbeckfondenR310-2018-3713 Denmark
Leducq Foundation17CVD05 France
Novo Nordisk FoundationNNF21OC0067647 Denmark
Novo Nordisk FoundationNNF17OC0029724 Denmark
Novo Nordisk FoundationNNF19OC0058439 Denmark
CitationJournal: EMBO J / Year: 2022
Title: Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.
Authors: Caroline Neumann / Lena Lindtoft Rosenbaek / Rasmus Kock Flygaard / Michael Habeck / Jesper Lykkegaard Karlsen / Yong Wang / Kresten Lindorff-Larsen / Hans Henrik Gad / Rune Hartmann / ...Authors: Caroline Neumann / Lena Lindtoft Rosenbaek / Rasmus Kock Flygaard / Michael Habeck / Jesper Lykkegaard Karlsen / Yong Wang / Kresten Lindorff-Larsen / Hans Henrik Gad / Rune Hartmann / Joseph Anthony Lyons / Robert A Fenton / Poul Nissen /
Abstract: The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na -dependent Cl - and K -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, ...The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na -dependent Cl - and K -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na , K , and 2 Cl ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl binding at the Cl1 site together with the nearby K ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.
History
DepositionApr 4, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14709.png

Downloads & links

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Map

FileDownload / File: emd_14709.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of human NKCC1 transmembrane domains
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 243.36 Å		1.01 Å/pix.
x 240 pix.
= 243.36 Å		1.01 Å/pix.
x 240 pix.
= 243.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.014 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.10764599 - 0.20131774
Average (Standard dev.)0.00015023017 (±0.0075521483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 243.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14709_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map, refined with a mask around transmembrane...

Fileemd_14709_additional_1.map
AnnotationFull map, refined with a mask around transmembrane domains, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened halfmap1

Fileemd_14709_half_map_1.map
AnnotationUnfiltered, unsharpened halfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened halfmap2

Fileemd_14709_half_map_2.map
AnnotationUnfiltered, unsharpened halfmap2
Projections & Slices
AxesZYX

Projections

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Histogram

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Sample components

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Entire : Dimeric NKCC1 in complex with Na+, K+ and 2Cl-

EntireName: Dimeric NKCC1 in complex with Na+, K+ and 2Cl-
Components
  • Complex: Dimeric NKCC1 in complex with Na+, K+ and 2Cl-
    • Complex: NKCC1 in complex with Na+, K+ and 2Cl-
      • Protein or peptide: Solute carrier family 12 member 2
    • Complex: NKCC1 in complex with Na+, K+ and 2Cl-
  • Ligand: SODIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water

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Supramolecule #1: Dimeric NKCC1 in complex with Na+, K+ and 2Cl-

SupramoleculeName: Dimeric NKCC1 in complex with Na+, K+ and 2Cl- / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 263.366 KDa

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Supramolecule #2: NKCC1 in complex with Na+, K+ and 2Cl-

SupramoleculeName: NKCC1 in complex with Na+, K+ and 2Cl- / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 131.683 KDa

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Supramolecule #3: NKCC1 in complex with Na+, K+ and 2Cl-

SupramoleculeName: NKCC1 in complex with Na+, K+ and 2Cl- / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 131.683 KDa

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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: 131.819625 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPAAAEPRPT APSSGAPGLA GVGETPSAAA LAAARVELPG TAVPSVPEDA APASRDGGGV RDEGPAAAGD GLGRPLGPTP SQSRFQVDL VSENAGRAAA AAAAAAAAAA AAGAGAGAKQ TPADGEASGE SEPAKGSEEA KGRFRVNFVD PAASSSAEDS L SDAAGVGV ...String:
GPAAAEPRPT APSSGAPGLA GVGETPSAAA LAAARVELPG TAVPSVPEDA APASRDGGGV RDEGPAAAGD GLGRPLGPTP SQSRFQVDL VSENAGRAAA AAAAAAAAAA AAGAGAGAKQ TPADGEASGE SEPAKGSEEA KGRFRVNFVD PAASSSAEDS L SDAAGVGV DGPNVSFQNG GDTVLSEGSS LHSGGGGGSG HHQHYYYDTH TNTYYLRTFG HNTMDAVPRI DHYRHTAAQL GE KLLRPSL AELHDELEKE PFEDGFANGE ESTPTRDAVV TYTAESKGVV KFGWIKGVLV RCMLNIWGVM LFIRLSWIVG QAG IGLSVL VIMMATVVTT ITGLSTSAIA TNGFVRGGGA YYLISRSLGP EFGGAIGLIF AFANAVAVAM YVVGFAETVV ELLK EHSIL MIDEINDIRI IGAITVVILL GISVAGMEWE AKAQIVLLVI LLLAIGDFVI GTFIPLESKK PKGFFGYKSE IFNEN FGPD FREEETFFSV FAIFFPAATG ILAGANISGD LADPQSAIPK GTLLAILITT LVYVGIAVSV GSCVVRDATG NVNDTI VTE LTNCTSAACK LNFDFSSCES SPCSYGLMNN FQVMSMVSGF TPLISAGIFS ATLSSALASL VSAPKIFQAL CKDNIYP AF QMFAKGYGKN NEPLRGYILT FLIALGFILI AELNVIAPII SNFFLASYAL INFSVFHASL AKSPGWRPAF KYYNMWIS L LGAILCCIVM FVINWWAALL TYVIVLGLYI YVTYKKPDVN WGSSTQALTY LNALQHSIRL SGVEDHVKNF RPQCLVMTG APNSRPALLH LVHDFTKNVG LMICGHVHMG PRRQAMKEMS IDQAKYQRWL IKNKMKAFYA PVHADDLREG AQYLMQAAGL GRMKPNTLV LGFKKDWLQA DMRDVDMYIN LFHDAFDIQY GVVVIRLKEG LDISHLQGQE ELLSSQEKSP GTKDVVVSVE Y SKKSDLDT SKPLSEKPIT HKVEEEDGKT ATQPLLKKES KGPIVPLNVA DQKLLEASTQ FQKKQGKNTI DVWWLFDDGG LT LLIPYLL TTKKKWKDCK IRVFIGGKIN RIDHDRRAMA TLLSKFRIDF SDIMVLGDIN TKPKKENIIA FEEIIEPYRL HED DKEQDI ADKMKEDEPW RITDNELELY KTKTYRQIRL NELLKEHSST ANIIVMSLPV ARKGAVSSAL YMAWLEALSK DLPP ILLVR GNHQSVLTFY S

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Macromolecule #2: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 2 / 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: 4 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #5: (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: 5 / 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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Macromolecule #6: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 6 / Number of copies: 6 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 24 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 8
Details: 0.01% glycol-diosgenin (GDN, Anatrace), 200mM NaCl, 200mM KCl, 20mM Tris-HCl pH 8.0 and 0.07mM bumetanide
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: GloQube (Quorum) 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Details: 3 uL sample was applied to the gold foil side of the grid and blotted iusing a blot force of 0 and blot time of 3-4 seconds before plunge- freezing into liquid ethane cooled by liquid nitrogen..
Detailsconcentration (mg/mL): 0.9-1.5

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number real images: 10063 / #0 - Average electron dose: 40.12 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number real images: 9122 / #1 - Average electron dose: 30.78 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingImage recording ID: 3 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 6190 / Average electron dose: 60.438 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Particle selectionNumber selected: 1002976
Details: Dataset 1: 1002976 particles Dataset 2: 1566691 Dataset 3: 2710603
Startup modelType of model: EMDB MAP
EMDB ID:

0473

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: RELION (ver. 3.1), cryoSPARC (ver. 3)) / Number images used: 258791
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6npl


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 61.44
Output model

PDB-7zgo:
Cryo-EM structure of human NKCC1 (TM domain)

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