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- PDB-9oc4: High-resolution cryo-EM structure of KdpFABC in the E1P-ADP state... -

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Basic information

Entry
Database: PDB / ID: 9oc4
TitleHigh-resolution cryo-EM structure of KdpFABC in the E1P-ADP state in lipid nanodisc
Components(Potassium-transporting ATPase ...) x 4
KeywordsTRANSPORT PROTEIN / P-type ATPase potassium channel transporter pump
Function / homology
Function and homology information


P-type K+ transporter / P-type potassium transmembrane transporter activity / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity ...P-type K+ transporter / P-type potassium transmembrane transporter activity / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Chem-9Y0 / ADENOSINE-5'-DIPHOSPHATE / : / Potassium-transporting ATPase potassium-binding subunit / Potassium-transporting ATPase ATP-binding subunit / Potassium-transporting ATPase KdpC subunit / Potassium-transporting ATPase KdpF subunit
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsHussein, A.K. / Zhang, X. / Pedersen, B.P. / Stokes, D.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM144109 United States
CitationJournal: bioRxiv / Year: 2025
Title: Conduction pathway for potassium through the E. coli pump KdpFABC.
Authors: Adel Hussein / Xihui Zhang / Bjørn Panyella Pedersen / David L Stokes /
Abstract: Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K pump to maintain intracellular potassium levels. The subunit KdpA belongs to ...Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K pump to maintain intracellular potassium levels. The subunit KdpA belongs to the Superfamily of K Transporters and adopts a pseudo-tetrameric architecture with a membrane embedded selectivity filter as seen in K channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work has hypothesized that K moves parallel to the membrane plane through a 40-Å long tunnel that connects the selectivity filter of KdpA with the binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 Å structure of the E1~P·ADP conformation, which reveals new features of the conduction pathway. This map shows exceedingly strong densities within the selectivity filter and at the canonical binding site, consistent with K bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K ions pass through the tunnel and support the existence of a low affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K gradient essential for bacterial survival.
History
DepositionApr 23, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 30, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium-transporting ATPase potassium-binding subunit
B: Potassium-transporting ATPase ATP-binding subunit
C: Potassium-transporting ATPase KdpC subunit
D: Potassium-transporting ATPase KdpF subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,92711
Polymers156,9374
Non-polymers1,9907
Water1,62190
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, part of a single operon
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Potassium-transporting ATPase ... , 4 types, 4 molecules ABCD

#1: Protein Potassium-transporting ATPase potassium-binding subunit / ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit A / Potassium-translocating ATPase A chain


Mass: 59218.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpA, b0698, JW0686 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P03959
#2: Protein Potassium-transporting ATPase ATP-binding subunit / ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit B / Potassium-translocating ATPase B chain


Mass: 72347.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpB, b0697, JW0685 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P03960, P-type K+ transporter
#3: Protein Potassium-transporting ATPase KdpC subunit / ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit C / Potassium-translocating ATPase C chain


Mass: 22299.225 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpC, b0696, JW0684 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P03961
#4: Protein/peptide Potassium-transporting ATPase KdpF subunit / ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit F / Potassium-translocating ATPase F chain


Mass: 3071.714 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpF, b4513, JW0687 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P36937

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Non-polymers , 5 types, 97 molecules

#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-9Y0 / (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C39H76NO8P
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 90 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: KdpFABC complex in the E1P-ADP state in lipid nanodisc
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.154150 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.4 / Details: 20 mM Tris pH 7.4, 100 mM KCl, and 0.5 mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2100 mMpotassium chlorideKCl1
30.5 mMTCEP1
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: EasyGlow / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 50000
EM imaging opticsEnergyfilter name: TFS Selectris

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2SerialEMimage acquisition
4cryoSPARC4.6.0CTF correction
7UCSF Chimera1.14model fitting
8Coot0.9.8.95model fitting
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
12cryoSPARC4.6.0classification
13cryoSPARC4.6.03D reconstruction
14PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10282000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 672405 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 59.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation
Atomic model buildingPDB-ID: 7LC3

7lc3


Accession code: 7LC3 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039372
ELECTRON MICROSCOPYf_angle_d0.47612746
ELECTRON MICROSCOPYf_dihedral_angle_d7.0271349
ELECTRON MICROSCOPYf_chiral_restr0.0351535
ELECTRON MICROSCOPYf_plane_restr0.0031586

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