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- EMDB-36220: Cryo-EM structure of Na+,K+-ATPase in the E1.Mg2+ state. -

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Basic information

Entry
Database: EMDB / ID: EMD-36220
TitleCryo-EM structure of Na+,K+-ATPase in the E1.Mg2+ state.
Map data
Sample
  • Complex: Na+,K+-ATPase
    • Protein or peptide: Na+,K+-ATPase beta subunit
    • Protein or peptide: Na, K-ATPase alpha subunit
    • Protein or peptide: FXYD domain-containing ion transport regulator
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsion pump / P-type ATPase / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKanai R / Vilsen B / Cornelius F / Toyoshima C
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19H00975 Japan
Japan Society for the Promotion of Science (JSPS)JP21K06109 Japan
CitationJournal: FEBS Lett / Year: 2023
Title: Crystal structures of Na ,K -ATPase reveal the mechanism that converts the K -bound form to Na -bound form and opens and closes the cytoplasmic gate.
Authors: Ryuta Kanai / Bente Vilsen / Flemming Cornelius / Chikashi Toyoshima /
Abstract: Na ,K -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na and K across the cell membrane by alternating between the E1 (showing high affinity for Na and low affinity ...Na ,K -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na and K across the cell membrane by alternating between the E1 (showing high affinity for Na and low affinity for K ) and E2 (low affinity to Na and high affinity to K ) forms. Presented here are two crystal structures of NKA in E1·Mg and E1·3Na states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K -bound E2·2K form to an E1 (E1·Mg ) form, which allows high-affinity Na binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na ) after binding three Na , while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.
History
DepositionMay 19, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36220.png

Downloads & links

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Map

FileDownload / File: emd_36220.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: 0.02
Minimum - Maximum-0.06552 - 0.1311914
Average (Standard dev.)0.00042294955 (±0.004975256)
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_36220_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36220_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 : Na+,K+-ATPase

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

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Supramolecule #1: Na+,K+-ATPase

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

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Macromolecule #1: Na+,K+-ATPase beta subunit

MacromoleculeName: Na+,K+-ATPase beta subunit / type: protein_or_peptide / ID: 1 / 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 #2: Na, K-ATPase alpha subunit

MacromoleculeName: Na, K-ATPase alpha subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Squalus acanthias (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

UniProtKB: Sodium/potassium-transporting ATPase subunit alpha

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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 / 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: 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
4.0 mMMgCl2magnesium chloride
30.0 mMC3H4N2imidazole
0.01 %C28H58O9C12E8
1.0 mMC4H10O2S2DTT
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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

Startup modelType of model: EMDB MAP
EMDB ID:

32900

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - 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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 36635
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8jbm


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

PDB-8jfz:
Cryo-EM structure of Na+,K+-ATPase in the E1.Mg2+ state.

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