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- PDB-9vj2: Crystal structure of palytoxin-bound Na+,K+-ATPase in the E2P state -

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Basic information

Entry
Database: PDB / ID: 9vj2
TitleCrystal structure of palytoxin-bound Na+,K+-ATPase in the E2P state
Components
  • (Sodium/potassium-transporting ATPase subunit ...) x 2
  • FXYD domain-containing ion transport regulator
KeywordsMEMBRANE PROTEIN / ion pump / P-type ATPase
Function / homology
Function and homology information


Basigin interactions / Ion homeostasis / P-type potassium transmembrane transporter activity / positive regulation of sodium ion export across plasma membrane / Ion transport by P-type ATPases / regulation of monoatomic ion transport / positive regulation of potassium ion import across plasma membrane / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization ...Basigin interactions / Ion homeostasis / P-type potassium transmembrane transporter activity / positive regulation of sodium ion export across plasma membrane / Ion transport by P-type ATPases / regulation of monoatomic ion transport / positive regulation of potassium ion import across plasma membrane / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization / establishment or maintenance of transmembrane electrochemical gradient / sodium ion export across plasma membrane / positive regulation of potassium ion transmembrane transport / intracellular sodium ion homeostasis / regulation of calcium ion transmembrane transport / ion channel regulator activity / relaxation of cardiac muscle / regulation of cardiac muscle contraction by calcium ion signaling / positive regulation of sodium ion transmembrane transport / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / ATPase activator activity / lateral plasma membrane / intercalated disc / sperm flagellum / transporter activator activity / ATP metabolic process / cardiac muscle contraction / T-tubule / proton transmembrane transport / protein localization to plasma membrane / sarcolemma / transmembrane transport / intracellular calcium ion homeostasis / melanosome / regulation of gene expression / ATPase binding / protein-macromolecule adaptor activity / basolateral plasma membrane / cell adhesion / protein stabilization / apical plasma membrane / axon / innate immune response / protein kinase binding / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
: / Ion-transport regulator, FXYD motif / : / ATP1G1/PLM/MAT8 family / FXYD family signature. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. ...: / Ion-transport regulator, FXYD motif / : / ATP1G1/PLM/MAT8 family / FXYD family signature. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / 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 / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
: / CHOLESTEROL / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Sodium/potassium-transporting ATPase subunit alpha / Sodium/potassium-transporting ATPase subunit beta-1 / FXYD domain-containing ion transport regulator
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.79 Å
AuthorsKanai, R. / Vilsen, B. / Cornelius, F. / Toyoshima, C.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP23K27136 Japan
Japan Society for the Promotion of Science (JSPS)JP24K01985 Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: How palytoxin transforms the Na,K pump into a cation channel.
Authors: Ryuta Kanai / Naoki Tsunekawa / Flemming Cornelius / Bente Vilsen / Chikashi Toyoshima /
Abstract: Palytoxin (PTX), a potent marine toxin, has long been known to transform Na,K-ATPase (NKA), an indispensable ion pump, into a nonselective cation channel. It has been postulated that PTX takes ...Palytoxin (PTX), a potent marine toxin, has long been known to transform Na,K-ATPase (NKA), an indispensable ion pump, into a nonselective cation channel. It has been postulated that PTX takes control of the two gates on either side of a channel-like pore. These gates normally open and close alternately, synchronized with chemical events, never opening simultaneously. A critical question is whether palytoxin takes over the control of the two gates or creates a new pathway. Here, we present structures of NKA with bound palytoxin in three different states. PTX binds to NKA in E2P, occupying the physiological Na exit pathway, similar to istaroxime, a new-generation cardiotonic steroid. Adding Na and ATP/ADP to the NKA·PTX complex induces an open channel traversing the entire membrane alongside the physiological ion pathway. As AlF, a stable transition state analog of phosphate replaces phosphate in the NKA·PTX complex preformed in E2P, the complex appears to undergo the normal reaction cycle from E2P to E1·Na. PTX occupies the space between the transmembrane helices M4 and M6, thereby preventing the closure of the extracellular half of the ion pathway. These structures demonstrate that the architecture of NKA is fundamentally different from "a pore with two gates." Each half of the ion pathway comprises three segments, including a movable component that plays a pivotal role in translocating the bound cations by connecting the constant part to an appropriate inlet. The ion pathway of NKA transforms dynamically, ensuring that the two halves never exist simultaneously.
History
DepositionJun 19, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium/potassium-transporting ATPase subunit alpha
B: Sodium/potassium-transporting ATPase subunit beta-1
G: FXYD domain-containing ion transport regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,10716
Polymers155,2363
Non-polymers8,87013
Water543
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10740 Å2
ΔGint-67 kcal/mol
Surface area60460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)85.022, 85.022, 646.129
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

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Sodium/potassium-transporting ATPase subunit ... , 2 types, 2 molecules AB

#1: Protein Sodium/potassium-transporting ATPase subunit alpha


Mass: 112937.766 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: D2WKD6
#2: Protein Sodium/potassium-transporting ATPase subunit beta-1 / Sodium/potassium-dependent ATPase subunit beta-1


Mass: 35204.402 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P05027

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Protein , 1 types, 1 molecules G

#3: Protein FXYD domain-containing ion transport regulator / Na+/K+ ATPase gamma subunit transcript variant a


Mass: 7094.115 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q58K79

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Sugars , 2 types, 2 molecules

#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#9: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 14 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#7: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#8: Chemical ChemComp-A1MA6 / palytoxin


Mass: 2680.139 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C129H223N3O54 / Feature type: SUBJECT OF INVESTIGATION
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.76 Å3/Da / Density % sol: 67.3 %
Crystal growTemperature: 288 K / Method: vapor diffusion, hanging drop / pH: 6.2
Details: 140 mM MgCl2, 25 mM NaCl, 15.4% (w/v) PEG2000MME, 7% (w/v) glycerol, 0.4 mM palytoxin, 1 mM BeCl3, 3.5 mM NaF, 5 mM GSH, 0.1 mM DTT and 1 mg/ml butylhydroxytoluen, 100 mM MES

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 18, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.79→50 Å / Num. obs: 11915 / % possible obs: 47.6 % / Redundancy: 12.7 % / CC1/2: 0.987 / Rmerge(I) obs: 0.259 / Net I/σ(I): 8.6
Reflection shellResolution: 3.79→4.25 Å / Redundancy: 7.2 % / Rmerge(I) obs: 1.307 / Mean I/σ(I) obs: 1.8 / Num. unique obs: 794 / CC1/2: 0.557 / % possible all: 11.1

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.79→10 Å / SU ML: 0.505 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 29.6975
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2757 519 5.01 %
Rwork0.2267 9847 -
obs0.2293 10366 44.14 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 134.52 Å2
Refinement stepCycle: LAST / Resolution: 3.79→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10378 0 416 3 10797
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.004811020
X-RAY DIFFRACTIONf_angle_d0.837114963
X-RAY DIFFRACTIONf_chiral_restr0.32881730
X-RAY DIFFRACTIONf_plane_restr0.00623189
X-RAY DIFFRACTIONf_dihedral_angle_d14.83454266
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.79-4.150.2559270.3394519X-RAY DIFFRACTION9.51
4.15-4.690.4017670.26951270X-RAY DIFFRACTION23.14
4.69-5.720.31721260.27022391X-RAY DIFFRACTION42.81
5.72-100.24922990.20365667X-RAY DIFFRACTION98.45

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