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

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Basic information

Entry
Database: PDB / ID: 9viy
TitleCryo-EM structure of palytoxin-bound Na+,K+-ATPase in the E2P state
Components
  • FXYD domain-containing ion transport regulator
  • Na+,K+-ATPase beta subunit
  • Sodium/potassium-transporting ATPase subunit alpha
KeywordsMEMBRANE PROTEIN / ion pump / P-type ATPase
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/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. ...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 / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
: / CHOLESTEROL / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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: Na+,K+-ATPase beta subunit
G: FXYD domain-containing ion transport regulator
C: Sodium/potassium-transporting ATPase subunit alpha
D: Na+,K+-ATPase beta subunit
E: FXYD domain-containing ion transport regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)348,79748
Polymers317,5246
Non-polymers31,27442
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C" or chain "D" or chain "E"
d_2ens_1chain "A" or chain "B" or chain "G"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ASPASPTYRTYRCD31 - 102336 - 1028
d_12CLRCLRCLRCLRCFA1301
d_13CLRCLRCLRCLRCGA1302
d_14PCWPCWPCWPCWCHA1304
d_15PCWPCWPCWPCWCIA1305
d_16PCWPCWPCWPCWCJA1306
d_17PCWPCWPCWPCWCKA1307
d_18PCWPCWPCWPCWCLA1308
d_19PCWPCWPCWPCWCMA1309
d_110PCWPCWPCWPCWCNA1310
d_111PCWPCWPCWPCWCOA1311
d_112PCWPCWPCWPCWCPA1312
d_113PCWPCWPCWPCWCQA1314
d_114MGMGMGMGCRA2003
d_115MGMGMGMGCSA2008
d_116PTXPTXPTXPTXCTA2011
d_117GLYGLYSERSERDE12 - 30512 - 305
d_118NAGNAGNAGNAGKK1
d_119NAGNAGNAGNAGKK2
d_120FUCFUCFUCFUCKK6
d_121BMABMABMABMAKK3
d_122MANMANMANMANKK4
d_123MANMANMANMANKK5
d_124NAGNAGNAGNAGLL1
d_125NAGNAGNAGNAGLL2
d_126BMABMABMABMALL3
d_127MANMANMANMANLL6
d_128MANMANMANMANLL4
d_129NAGNAGNAGNAGLL5
d_130NAGNAGNAGNAGMM1
d_131NAGNAGNAGNAGMM2
d_132FUCFUCFUCFUCMM5
d_133BMABMABMABMAMM3
d_134MANMANMANMANMM4
d_135NAGNAGNAGNAGNN1
d_136NAGNAGNAGNAGNN2
d_137CLRCLRCLRCLRDUA501
d_138CLRCLRCLRCLRDVA502
d_139GLUGLUCYSCYSEF4 - 4324 - 63
d_21ASPASPTYRTYRAA31 - 102336 - 1028
d_22CLRCLRCLRCLRAO1301
d_23CLRCLRCLRCLRAP1302
d_24PCWPCWPCWPCWAQ1304
d_25PCWPCWPCWPCWAR1305
d_26PCWPCWPCWPCWAS1306
d_27PCWPCWPCWPCWAT1307
d_28PCWPCWPCWPCWAU1308
d_29PCWPCWPCWPCWAV1309
d_210PCWPCWPCWPCWAW1310
d_211PCWPCWPCWPCWAX1311
d_212PCWPCWPCWPCWAY1312
d_213PCWPCWPCWPCWAZ1314
d_214MGMGMGMGAAA2003
d_215MGMGMGMGABA2008
d_216PTXPTXPTXPTXACA2011
d_217GLYGLYSERSERBB12 - 30512 - 305
d_218NAGNAGNAGNAGFG1
d_219NAGNAGNAGNAGFG2
d_220FUCFUCFUCFUCFG6
d_221BMABMABMABMAFG3
d_222MANMANMANMANFG4
d_223MANMANMANMANFG5
d_224NAGNAGNAGNAGHH1
d_225NAGNAGNAGNAGHH2
d_226BMABMABMABMAHH3
d_227MANMANMANMANHH6
d_228MANMANMANMANHH4
d_229NAGNAGNAGNAGHH5
d_230NAGNAGNAGNAGII1
d_231NAGNAGNAGNAGII2
d_232FUCFUCFUCFUCII5
d_233BMABMABMABMAII3
d_234MANMANMANMANII4
d_235NAGNAGNAGNAGJJ1
d_236NAGNAGNAGNAGJJ2
d_237CLRCLRCLRCLRBDA501
d_238CLRCLRCLRCLRBEA502
d_239GLUGLUCYSCYSGC4 - 4324 - 63

NCS oper: (Code: givenMatrix: (-0.999981037163, -0.00252572818611, 0.00561658364052), (0.00254760001627, -0.999989187151, 0.00389041313211), (0.00560669678315, 0.00390464816741, 0.999976659065)Vector: ...NCS oper: (Code: given
Matrix: (-0.999981037163, -0.00252572818611, 0.00561658364052), (0.00254760001627, -0.999989187151, 0.00389041313211), (0.00560669678315, 0.00390464816741, 0.999976659065)
Vector: 257.730905272, 257.479173103, -0.989643580003)

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Components

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Protein , 3 types, 6 molecules ACBDGE

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


Mass: 113389.859 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: Q4H132
#2: Protein Na+,K+-ATPase beta subunit / Sodium/potassium-transporting ATPase subunit beta-1


Mass: 35176.125 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: C4IX13
#3: Protein FXYD domain-containing ion transport regulator


Mass: 10195.847 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: Q70Q12

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Sugars , 4 types, 8 molecules

#4: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1114.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-1-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][b-D-GlcpNAc]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 894.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/4,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-4/a4-b1_a6-e1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#7: 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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 34 molecules

#8: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C27H46O
#9: 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.: 20 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-A1MA6 / palytoxin


Mass: 2680.139 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C129H223N3O54 / Feature type: SUBJECT OF INVESTIGATION

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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: Na+,K+-ATPase / Type: COMPLEX / Entity ID: #2, #1, #3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Squalus acanthias (spiny dogfish)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
11 mMmagnesium chlorideMgCl21
220 mMHEPESC8H18N2O4S1
30.01 %C12E8C28H58O91
41 mMDTTC4H10O2S21
50.3 mMphosphateHPO41
60.4 mMpalytoxinC129H223N3O541
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99.9 % / Chamber temperature: 279 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: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION4.0.1particle selection
2PHENIX1.21.2_5419model refinement
5CTFFIND4.1.14CTF correction
10RELION4.0.1initial Euler assignment
11RELION4.0.1final Euler assignment
12RELION4.0.1classification
13RELION4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24807 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7wz0

7wz0


Accession code: 7wz0 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.89 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006323332
ELECTRON MICROSCOPYf_angle_d1.235431502
ELECTRON MICROSCOPYf_chiral_restr0.23213684
ELECTRON MICROSCOPYf_plane_restr0.00746466
ELECTRON MICROSCOPYf_dihedral_angle_d19.56999698
Refine LS restraints NCSType: NCS constraints / Rms dev position: 3.35904945387E-13 Å

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