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- EMDB-65098: Cryo-EM structure of palytoxin-bound Na+,K+-ATPase in the E2P state -

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Basic information

Entry
Database: EMDB / ID: EMD-65098
TitleCryo-EM structure of palytoxin-bound Na+,K+-ATPase in the E2P state
Map data
Sample
  • Complex: Na+,K+-ATPase
    • Protein or peptide: Na+,K+-ATPase beta subunit
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: FXYD domain-containing ion transport regulator
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: MAGNESIUM ION
  • Ligand: palytoxin
Keywordsion pump / P-type ATPase / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of monoatomic ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / ATPase activator activity / sodium channel regulator activity / monoatomic ion transport ...regulation of monoatomic ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / ATPase activator activity / sodium channel regulator activity / monoatomic ion transport / proton transmembrane transport / ATP hydrolysis activity / ATP binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
Ion-transport regulator, FXYD motif / : / ATP1G1/PLM/MAT8 family / FXYD family signature. / Sodium and potassium ATPases beta subunits signature 2. / 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 and potassium ATPases beta subunits signature 2. / 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. / : / 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 / P-type ATPase, cytoplasmic domain N / P-type ATPase actuator domain / 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
Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit alpha / FXYD domain-containing ion transport regulator
Similarity search - Component
Biological speciesSqualus acanthias (spiny dogfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKanai R / Vilsen B / Cornelius F / Toyoshima C
Funding support Japan, 2 items
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, 2025-
Header (metadata) releaseOct 1, 2025-
Map releaseOct 1, 2025-
UpdateOct 1, 2025-
Current statusOct 1, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_65098.png

Downloads & links

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Map

FileDownload / File: emd_65098.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 240 pix.
= 258.24 Å		1.08 Å/pix.
x 240 pix.
= 258.24 Å		1.08 Å/pix.
x 240 pix.
= 258.24 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0095
Minimum - Maximum-0.029217156 - 0.063411355
Average (Standard dev.)0.00027313823 (±0.002042432)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 258.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_65098_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_65098_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Na+,K+-ATPase

EntireName: Na+,K+-ATPase
Components
  • Complex: Na+,K+-ATPase
    • Protein or peptide: Na+,K+-ATPase beta subunit
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: FXYD domain-containing ion transport regulator
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: MAGNESIUM ION
  • Ligand: palytoxin

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Supramolecule #1: Na+,K+-ATPase

SupramoleculeName: Na+,K+-ATPase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1, #3
Source (natural)Organism: Squalus acanthias (spiny dogfish)

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

MacromoleculeName: Sodium/potassium-transporting ATPase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Squalus acanthias (spiny dogfish)
Molecular weightTheoretical: 113.389859 KDa
SequenceString: MGKGTASDKY EPAATSENAT KSKKKGKKDK IDKKRDLDEL KKEVSMDDHK LSLDELHNKY GTDLTRGLTN ARAKEILARD GPNSLTPPP TTPEWIKFCR QLFGGFSILL WIGAILCFLA YGIQAATEDE PANDNLYLGV VLSTVVIVTG CFSYYQEAKS S RIMDSFKN ...String:
MGKGTASDKY EPAATSENAT KSKKKGKKDK IDKKRDLDEL KKEVSMDDHK LSLDELHNKY GTDLTRGLTN ARAKEILARD GPNSLTPPP TTPEWIKFCR QLFGGFSILL WIGAILCFLA YGIQAATEDE PANDNLYLGV VLSTVVIVTG CFSYYQEAKS S RIMDSFKN MVPQQALVIR DGEKSTINAE FVVAGDLVEV KGGDRIPADL RIISAHGCKV DNSSLTGESE PQTRSPEFSS EN PLETRNI AFFSTNCVEG TARGVVVYTG DRTVMGRIAT LASGLEVGRT PIAIEIEHFI HIITGVAVFL GVSFFILSLI LGY SWLEAV IFLIGIIVAN VPEGLLATVT VCLTLTAKRM ARKNCLVKNL EAVETLGSTS TICS(PHD)KTGTL TQNRMTVAHM WFDNQIHEA DTTENQSGAA FDKTSATWSA LSRIAALCNR AVFQAGQDNV PILKRSVAGD ASESALLKCI ELCCGSVQGM R DRNPKIVE IPFNSTNKYQ LSIHENEKSS ESRYLLVMKG APERILDRCS TILLNGAEEP LKEDMKEAFQ NAYLELGGLG ER VLGFCHF ALPEDKYNEG YPFDADEPNF PTTDLCFVGL MAMIDPPRAA VPDAVGKCRS AGIKVIMVTG DHPITAKAIA KGV GIISEG NETIEDIAAR LNIPIGQVNP RDAKACVVHG SDLKDLSTEV LDDILHYHTE IVFARTSPQQ KLIIVEGCQR QGAI VAVTG DGVNDSPALK KADIGVAMGI SGSDVSKQAA DMILLDDNFA SIVTGVEEGR LIFDNLKKSI AYTLTSNIPE ITPFL VFII GNVPLPLGTV TILCIDLGTD MVPAISLAYE QAESDIMKRQ PRNPKTDKLV NERLISMAYG QIGMIQALGG FFSYFV ILA ENGFLPMDLI GKRVRWDDRW ISDVEDSFGQ QWTYEQRKIV EFTCHTSFFI SIVVVQWADL IICKTRRNSI FQQGMKN KI LIFGLFEETA LAAFLSYCPG TDVALRMYPL KPSWWFCAFP YSLIIFLYDE MRRFIIRRSP GGWVEQETYY

UniProtKB: Sodium/potassium-transporting ATPase subunit alpha

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Macromolecule #2: Na+,K+-ATPase beta subunit

MacromoleculeName: Na+,K+-ATPase beta subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Squalus acanthias (spiny dogfish)
Molecular weightTheoretical: 35.176125 KDa
SequenceString: MARGKSKETD GGWKKFLWDS EKKEFLGRTG SSWFKIFLFY LIFYGCLAGI FIGTIQVLLL TLSDFEPKYQ DRVAPPGLSH APYAIKTEI SFSISNPKSY ESFVKSMHKL MDLYNESSQA GNSPFEDCSD TPADYIKRGD LDDSQGQKKA CRFSRMWLKN C SGLDDTTY ...String:
MARGKSKETD GGWKKFLWDS EKKEFLGRTG SSWFKIFLFY LIFYGCLAGI FIGTIQVLLL TLSDFEPKYQ DRVAPPGLSH APYAIKTEI SFSISNPKSY ESFVKSMHKL MDLYNESSQA GNSPFEDCSD TPADYIKRGD LDDSQGQKKA CRFSRMWLKN C SGLDDTTY GYAEGKPCVV AKLNRIIGFY PKPLKNTTDL PEELQANYNQ YVLPLRCAAK REEDREKIGS IEYFGLGGYA GF PLQYYPY YGKRLQKKYL QPLLAIQFTN LTQNMELRIE CKVYGENIDY SEKDRFRGRF EVKIEVKS

UniProtKB: Sodium/potassium-transporting ATPase subunit beta

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Macromolecule #3: FXYD domain-containing ion transport regulator

MacromoleculeName: FXYD domain-containing ion transport regulator / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Squalus acanthias (spiny dogfish)
Molecular weightTheoretical: 10.195847 KDa
SequenceString:
MLGAATGLMV LVAVTQGVWA MDPEGPDNDE RFTYDYYRLR VVGLIVAAVL CVIGIIILLA GKCRCKFNQN KRTRSNSGTA TAQHLLQPG EATEC

UniProtKB: FXYD domain-containing ion transport regulator

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Macromolecule #8: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 8 / Number of copies: 8 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #9: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 9 / Number of copies: 20 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: palytoxin

MacromoleculeName: palytoxin / type: ligand / ID: 11 / Number of copies: 2 / Formula: A1MA6
Molecular weightTheoretical: 2.680139 KDa

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
1.0 mMMgCl2magnesium chloride
20.0 mMC8H18N2O4SHEPES
0.01 %C28H58O9C12E8
1.0 mMC4H10O2S2DTT
0.3 mMHPO4phosphate
0.4 mMC129H223N3O54palytoxin
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 99.9 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

32900

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0.1) / Number images used: 24807
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7wz0


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9viy:
Cryo-EM structure of palytoxin-bound Na+,K+-ATPase in the E2P state

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