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- EMDB-33601: Cryo-EM structure of the Na+,K+-ATPase in the E2.2K+ state -

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Basic information

Entry
Database: EMDB / ID: EMD-33601
TitleCryo-EM structure of the Na+,K+-ATPase in the E2.2K+ state
Map data
Sample
  • Complex: Sodium/potassium-transporting ATPase
    • Protein or peptide: Na, K-ATPase alpha subunit
    • Protein or peptide: Na+,K+-ATPase beta subunit
    • Protein or peptide: FXYD domain-containing ion transport regulator
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water
Function / homology
Function and homology information


regulation of monoatomic ion transport / P-type potassium transmembrane transporter activity / sodium:potassium-exchanging ATPase complex / ion channel regulator activity / sodium ion transport / monoatomic ion transport / potassium ion transport / ATP hydrolysis activity / ATP binding / membrane ...regulation of monoatomic ion transport / P-type potassium transmembrane transporter activity / sodium:potassium-exchanging ATPase complex / ion channel regulator activity / sodium ion transport / monoatomic ion transport / potassium ion transport / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / 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. / P-type ATPase subfamily IIC, subunit alpha ...: / 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
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) / spiny dogfish (spiny dogfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKanai R / Cornelius F / Vilsen B / Toyoshima C
Funding support Japan, Denmark, 6 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H00975 Japan
Japan Society for the Promotion of Science (JSPS)18K06148 Japan
Danish Council for Independent ResearchDFF7016-00193B Denmark
Danish Council for Independent ResearchDFF4183-00011A Denmark
Novo Nordisk FoundationNNF14OC0013409 Denmark
Novo Nordisk FoundationNNF13OC0006555 Denmark
CitationJournal: FEBS Lett / Year: 2022
Title: Cryo-electron microscopy of Na ,K -ATPase reveals how the extracellular gate locks in the E2·2K state.
Authors: Ryuta Kanai / Flemming Cornelius / Bente Vilsen / Chikashi Toyoshima /
Abstract: Na ,K -ATPase (NKA) is one of the most important members of the P-type ion-translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na and K across the cell ...Na ,K -ATPase (NKA) is one of the most important members of the P-type ion-translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na and K across the cell membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K state solved by cryo-electron microscopy. It is a stable state with two occluded K after transferring three Na into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K , linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K into the cytoplasm, more than 100 times.
History
DepositionJun 14, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33601.png

Downloads & links

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Map

FileDownload / File: emd_33601.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.076 Å
Density
Contour LevelBy AUTHOR: 0.0199
Minimum - Maximum-0.057013754 - 0.12149854
Average (Standard dev.)0.0005845816 (±0.0049232873)
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: #1

Fileemd_33601_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33601_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 : Sodium/potassium-transporting ATPase

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

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Supramolecule #1: Sodium/potassium-transporting ATPase

SupramoleculeName: Sodium/potassium-transporting ATPase / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Squalus acanthias (spiny dogfish)

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Macromolecule #1: Na, K-ATPase alpha subunit

MacromoleculeName: Na, K-ATPase alpha subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spiny dogfish (spiny dogfish)
Molecular weightTheoretical: 113.309891 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 TICSDKTGTL TQNRMTVAHM WFDN QIHEA DTTENQSGAA FDKTSATWSA LSRIAALCNR AVFQAGQDNV PILKRSVAGD ASESALLKCI ELCCGSVQGM RDRNP KIVE IPFNSTNKYQ LSIHENEKSS ESRYLLVMKG APERILDRCS TILLNGAEEP LKEDMKEAFQ NAYLELGGLG ERVLGF CHF ALPEDKYNEG YPFDADEPNF PTTDLCFVGL MAMIDPPRAA VPDAVGKCRS AGIKVIMVTG DHPITAKAIA KGVGIIS EG NETIEDIAAR LNIPIGQVNP RDAKACVVHG SDLKDLSTEV LDDILHYHTE IVFARTSPQQ KLIIVEGCQR QGAIVAVT G DGVNDSPALK KADIGVAMGI SGSDVSKQAA DMILLDDNFA SIVTGVEEGR LIFDNLKKSI AYTLTSNIPE ITPFLVFII GNVPLPLGTV TILCIDLGTD MVPAISLAYE QAESDIMKRQ PRNPKTDKLV NERLISMAYG QIGMIQALGG FFSYFVILAE NGFLPMDLI GKRVRWDDRW ISDVEDSFGQ QWTYEQRKIV EFTCHTSFFI SIVVVQWADL IICKTRRNSI FQQGMKNKIL I FGLFEETA LAAFLSYCPG TDVALRMYPL KPSWWFCAFP YSLIIFLYDE MRRFIIRRSP GGWVEQETYY

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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: spiny dogfish (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

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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: spiny dogfish (spiny dogfish)
Molecular weightTheoretical: 10.195847 KDa
SequenceString:
MLGAATGLMV LVAVTQGVWA MDPEGPDNDE RFTYDYYRLR VVGLIVAAVL CVIGIIILLA GKCRCKFNQN KRTRSNSGTA TAQHLLQPG EATEC

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Macromolecule #8: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 8 / Number of copies: 6 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #9: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 9 / Number of copies: 8 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #10: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 10 / Number of copies: 26 / 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 #11: water

MacromoleculeName: water / type: ligand / ID: 11 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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
100.0 mMKClpotassium chloride
3.0 mMMgCl2magnesium chloride
20.0 mMC3H4N2imidazole
1.0 mMC4H10O2S2DTT
0.01 %C28H58O9C12E8
GridModel: C-flat-1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14)
Startup modelType of model: OTHER / Details: PDBcode: 7WYW was used as the startup model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
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. 3.1.3) / Number images used: 47461
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2zxe

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 86 / Target criteria: Correlation Coeeficient
Output model

PDB-7y45:
Cryo-EM structure of the Na+,K+-ATPase in the E2.2K+ state

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