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- EMDB-16319: Structure of the K+/H+ exchanger KefC with GSH -

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Basic information

Entry
Database: EMDB / ID: EMD-16319
TitleStructure of the K+/H+ exchanger KefC with GSH
Map data
Sample
  • Complex: Kefc protein dimer with GSH
    • Protein or peptide: Glutathione-regulated potassium-efflux system protein KefC
  • Ligand: POTASSIUM ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: GLUTATHIONE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
Keywordspotassium proton exchanger / KefC / Transporter / CPA / MEMBRANE PROTEIN / GSH
Function / homology
Function and homology information


glutathione-regulated potassium exporter activity / response to methylglyoxal / antiporter activity / toxic substance binding / proton transmembrane transport / enzyme binding / plasma membrane
Similarity search - Function
Glutathione-regulated potassium-efflux system protein KefC / K+/H+ exchanger / Potassium uptake protein TrkA / Sodium/solute symporter superfamily / Cation/H+ exchanger / Sodium/hydrogen exchanger family / Regulator of K+ conductance, N-terminal / TrkA-N domain / RCK N-terminal domain profile. / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Glutathione-regulated potassium-efflux system protein KefC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsGulati A / Drew D
Funding supportEuropean Union, Sweden, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-CoG-820187European Union
Swedish Research Council Sweden
CitationJournal: Nat Commun / Year: 2024
Title: Structure and mechanism of the K/H exchanger KefC.
Authors: Ashutosh Gulati / Surabhi Kokane / Annemarie Perez-Boerema / Claudia Alleva / Pascal F Meier / Rei Matsuoka / David Drew /
Abstract: Intracellular potassium (K) homeostasis is fundamental to cell viability. In addition to channels, K levels are maintained by various ion transporters. One major family is the proton-driven K efflux ...Intracellular potassium (K) homeostasis is fundamental to cell viability. In addition to channels, K levels are maintained by various ion transporters. One major family is the proton-driven K efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na/H antiporter NapA. By structural assignment of a coordinated K ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K ion. KefC harbors C-terminal regulator of K conductance (RCK) domains, as present in some bacterial K-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K-ion-channels.
History
DepositionDec 11, 2022-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16319.png

Downloads & links

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Map

FileDownload / File: emd_16319.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.89 Å/pix.
x 300 pix.
= 265.8 Å		0.89 Å/pix.
x 300 pix.
= 265.8 Å		0.89 Å/pix.
x 300 pix.
= 265.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.886 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.47265485 - 0.92246854
Average (Standard dev.)0.00038364407 (±0.016745644)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 265.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16319_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16319_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Kefc protein dimer with GSH

EntireName: Kefc protein dimer with GSH
Components
  • Complex: Kefc protein dimer with GSH
    • Protein or peptide: Glutathione-regulated potassium-efflux system protein KefC
  • Ligand: POTASSIUM ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: GLUTATHIONE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: Kefc protein dimer with GSH

SupramoleculeName: Kefc protein dimer with GSH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Glutathione-regulated potassium-efflux system protein KefC

MacromoleculeName: Glutathione-regulated potassium-efflux system protein KefC
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 61.183223 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDSHTLIQAL IYLGSAALIV PIAVRLGLGS VLGYLIAGCI IGPWGLRLVT DAESILHFAE IGVVLMLFII GLELDPQRLW KLRAAVFGG GALQMVICGG LLGLFCMLLG LRWQVAELIG MTLALSSTAI AMQAMNERNL MVTQMGRSAF AVLLFQDIAA I PLVAMIPL ...String:
MDSHTLIQAL IYLGSAALIV PIAVRLGLGS VLGYLIAGCI IGPWGLRLVT DAESILHFAE IGVVLMLFII GLELDPQRLW KLRAAVFGG GALQMVICGG LLGLFCMLLG LRWQVAELIG MTLALSSTAI AMQAMNERNL MVTQMGRSAF AVLLFQDIAA I PLVAMIPL LATSSASTTM GAFALSALKV AGALVLVVLL GRYVTRPALR FVARSGLREV FSAVALFLVF GFGLLLEEVG LS MAMGAFL AGVLLASSEY RHALESDIEP FKGLLLGLFF IGVGMSIDFG TLLENPLRIV ILLLGFLIIK IAMLWLIARP LQV PNKQRR WFAVLLGQGS EFAFVVFGAA QMANVLEPEW AKSLTLAVAL SMAATPILLV ILNRLEQSST EEAREADEID EEQP RVIIA GFGRFGQITG RLLLSSGVKM VVLDHDPDHI ETLRKFGMKV FYGDATRMDL LESAGAAKAE VLINAIDDPQ TNLQL TEMV KEHFPHLQII ARARDVDHYI RLRQAGVEKP ERETFEGALK TGRLALESLG LGPYEARERA DVFRRFNIQM VEEMAM V

UniProtKB: Glutathione-regulated potassium-efflux system protein KefC

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #3: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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Macromolecule #4: GLUTATHIONE

MacromoleculeName: GLUTATHIONE / type: ligand / ID: 4 / Number of copies: 2 / Formula: GSH
Molecular weightTheoretical: 307.323 Da
Chemical component information

ChemComp-GSH:
GLUTATHIONE

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Macromolecule #5: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 5 / Number of copies: 2 / Formula: PGW
Molecular weightTheoretical: 749.007 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

BufferpH: 7.5
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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 64.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 260115
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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