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- EMDB-36794: Cryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-36794
TitleCryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in cation-free E2P form
Map data
Sample
  • Complex: Na+,K+-ATPase alpha2/beta2 from Artemia Salina
    • Protein or peptide: Na+,K+-ATPase alpha2KK
    • Protein or peptide: Na+,K+-ATPase beta2
  • Ligand: TETRAFLUOROALUMINATE ION
KeywordsP-type ATPase / sodium pump / membrane protein / transporter / TRANSPORT PROTEIN
Biological speciesArtemia salina (crustacean)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsAbe K / Artigas P
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.
Authors: Pablo Artigas / Dylan J Meyer / Victoria C Young / Kerri Spontarelli / Jessica Eastman / Evan Strandquist / Huan Rui / Benoît Roux / Matthew A Birk / Hanayo Nakanishi / Kazuhiro Abe / Craig Gatto /
Abstract: Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange ...Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange for 2 K per hydrolyzed ATP. express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines ( α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that express two salinity-independent canonical α subunits (α1 and α3), as well as two β variants, in addition to the salinity-controlled α2. These β subunits permitted heterologous expression of the α2 pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2 pumps and compared it to that of α1 (and its α2-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2-like characteristics to α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2's Lys308 helps to maintain high affinity for external K when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K-congener Rb, we prove that double-lysine-substituted pumps transport 2Na and 1 K per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing to maintain steeper Na gradients in hypersaline environments.
History
DepositionJul 11, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36794.png

Downloads & links

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Map

FileDownload / File: emd_36794.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.752 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.0031608 - 1.8978219
Average (Standard dev.)-0.0015046176 (±0.036503237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 338.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36794_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_36794_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_36794_half_map_2.map
Projections & Slices
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Sample components

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Entire : Na+,K+-ATPase alpha2/beta2 from Artemia Salina

EntireName: Na+,K+-ATPase alpha2/beta2 from Artemia Salina
Components
  • Complex: Na+,K+-ATPase alpha2/beta2 from Artemia Salina
    • Protein or peptide: Na+,K+-ATPase alpha2KK
    • Protein or peptide: Na+,K+-ATPase beta2
  • Ligand: TETRAFLUOROALUMINATE ION

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Supramolecule #1: Na+,K+-ATPase alpha2/beta2 from Artemia Salina

SupramoleculeName: Na+,K+-ATPase alpha2/beta2 from Artemia Salina / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Artemia salina (crustacean)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Na+,K+-ATPase alpha2KK

MacromoleculeName: Na+,K+-ATPase alpha2KK / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Artemia salina (crustacean)
Molecular weightTheoretical: 111.144211 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGKKQGKQLS DLKKELELDQ HKIPLEELCR RLGTNTETGL TSSQAKSHLE KYGPNALTPP RTTPEWIKFC KQLFGGFQML LWIGSILCF IAYTMEKYKN PDVLGDNLYL GLALLFVVIM TGCFAYYQDH NASKIMDSFK NLMPQFAFVI RDGKKIQLKA E EVTVGDLV ...String:
MGKKQGKQLS DLKKELELDQ HKIPLEELCR RLGTNTETGL TSSQAKSHLE KYGPNALTPP RTTPEWIKFC KQLFGGFQML LWIGSILCF IAYTMEKYKN PDVLGDNLYL GLALLFVVIM TGCFAYYQDH NASKIMDSFK NLMPQFAFVI RDGKKIQLKA E EVTVGDLV EVKFGDRIPA DIRITSCQSM KVDNSSLTGE SEPQSRSTEC TNDNPLETKN LAFFFTNTLE GTGRGIVINV GD DSVMGRI ACLASSLDSG KTPIAREIEH FIHIITAMAV SLAAVFAVIS FLYGYTWLEA AIFMIGIIVA KVPEGLLATV TVC LTLTAK RMAKKNCLVR NLEAVETLGS TSTICSDKTG TLTQNRMTVA HMWFDQKIVT ADTTENQSGN QLYRGSKGFP ELIR VASLC SRAEFKTEHA HLPVLKRDVN GDASEAAILK FAEMSTGSVM NIRSKQKKVS EIPFNSANKY QVSVHEREDK SGYFL VMKG APERILERCS TILIDGTEIP LDNHMKECFN NAYMELGGMG ERVLGFCDFE LPSDQYPRGY VFDADEPNFP ISGLRF VGL MSMIDPPRAA VPDAVSKCRS AGIKVIMVTG DHPITAKAIA RQVGIISEGH ETVDDIAARL NIPVSEVNPR SAQAAVI HG NDLKDMNSDQ LDDILRHYRE IVFARTSPQQ KLIIVEGVQR QGEFVAVTGD GVNDSPALKK ADIGVAMGIA GSDVSKQA A DMILLDDNFA SIVTGVEEGR LIFDNIKKSI AYTLTSKIPE LSPFLMYILF DLPLAIGTVT ILCIDLGTDV VPAISMAYE GPEADPRKPR DPVKEKLVNE RLISMAYGQI GVMQAFGGFF TYFVIMGECG FLPNRLFGLR KWWESKAYND LTDSYGQEWT WDARKQLEY TCHTAFFISI VIVQWTDLII CKTRRLSLFQ QGMKNGTLNF ALVFETCVAA FLSYTPGMDK GLRMYPLKIW W WFPPMPFS LLILVYDECR KFLMRRNPGG FLERETYY

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Macromolecule #2: Na+,K+-ATPase beta2

MacromoleculeName: Na+,K+-ATPase beta2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Artemia salina (crustacean)
Molecular weightTheoretical: 38.496422 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGDYKDDDDK SSGENLYFQG MADKKPEEFF VGSGPKPTKW QSVKTFIWNS ETSEFMGRTG VNWAKITIFY VIFYTLLAGF FAGMLMIFY QTLDFKIPKW QNKDSLIGTN PGLGFRPMPP EAQVDSTLIQ FKHGIKGDWQ YWVHSLTEFL EPYETLTSSG Q EFTNCDFD ...String:
MGDYKDDDDK SSGENLYFQG MADKKPEEFF VGSGPKPTKW QSVKTFIWNS ETSEFMGRTG VNWAKITIFY VIFYTLLAGF FAGMLMIFY QTLDFKIPKW QNKDSLIGTN PGLGFRPMPP EAQVDSTLIQ FKHGIKGDWQ YWVHSLTEFL EPYETLTSSG Q EFTNCDFD KPPQEGKACN FNVELLGDHC TKENNFGYEL GKPCVLIKLN KIFGWRPEVY NSSAEVPEDM PADLKSYIKD IE TGNKTHM NMVWLSCEGE TANDKEKIGT ITYTPFRGFP AYYYPYLNVP GYLTPVVALQ FGSLQNGQAV NVECKAWANN ISR DRQRRL GSVHFEIRMD

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Macromolecule #3: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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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

Concentration8 mg/mL
BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2zxe

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39483
FSC plot (resolution estimation)

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