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- EMDB-32957: Cryo-EM structure of non gastric H,K-ATPase alpha2 SPWC mutant in... -

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Basic information

Entry
Database: EMDB / ID: EMD-32957
TitleCryo-EM structure of non gastric H,K-ATPase alpha2 SPWC mutant in (2K+)E2-AlF state
Map data
Sample
  • Complex: non gastric H,K-ATPase
    • Protein or peptide: Potassium-transporting ATPase alpha chain 2
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit beta-1
  • Ligand: POTASSIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CHOLESTEROL
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Basigin interactions / H+/K+-exchanging ATPase / Ion transport by P-type ATPases / Na+/K+-exchanging ATPase / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex ...Basigin interactions / H+/K+-exchanging ATPase / Ion transport by P-type ATPases / Na+/K+-exchanging ATPase / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization / regulation of pH / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / regulation of calcium ion transmembrane transport / potassium ion homeostasis / intracellular sodium ion homeostasis / regulation of cardiac muscle contraction by calcium ion signaling / response to metal ion / relaxation of cardiac muscle / Ion homeostasis / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / monoatomic cation transmembrane transport / ATPase activator activity / blastocyst development / intercalated disc / lateral plasma membrane / sperm flagellum / sodium ion transmembrane transport / cardiac muscle contraction / ATP metabolic process / T-tubule / proton transmembrane transport / protein localization to plasma membrane / potassium ion transport / caveola / response to organic cyclic compound / sarcolemma / intracellular calcium ion homeostasis / ATPase binding / protein-macromolecule adaptor activity / regulation of gene expression / basolateral plasma membrane / response to hypoxia / cell adhesion / protein stabilization / protein heterodimerization activity / apical plasma membrane / innate immune response / protein kinase binding / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
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 ...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-1 / Potassium-transporting ATPase alpha chain 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAbe K / Nakanishi H / Young V / Artigas P
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structure and function of H/K pump mutants reveal Na/K pump mechanisms.
Authors: Victoria C Young / Hanayo Nakanishi / Dylan J Meyer / Tomohiro Nishizawa / Atsunori Oshima / Pablo Artigas / Kazuhiro Abe /
Abstract: Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na to H selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via ...Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na to H selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H/K pump, a strict H-dependent electroneutral P-type ATPase, into a bona fide Na-dependent electrogenic Na/K pump. Conversion of a H-dependent primary-active transporter into a Na-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H/K pump, a suitable drug target to treat cystic fibrosis, and of its Na/K pump-mimicking mutant in two major conformations, providing insight on how Na binding drives a concerted mechanism leading to Na/K pump phosphorylation.
History
DepositionFeb 25, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32957.png

Downloads & links

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Map

FileDownload / File: emd_32957.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0216
Minimum - Maximum-0.08744202 - 0.14196795
Average (Standard dev.)0.00037650246 (±0.0041901404)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32957_msk_1.map
Projections & Slices
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Density Histograms

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Histogram (log scale)

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Half map: #1

Fileemd_32957_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32957_half_map_2.map
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Sample components

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Entire : non gastric H,K-ATPase

EntireName: non gastric H,K-ATPase
Components
  • Complex: non gastric H,K-ATPase
    • Protein or peptide: Potassium-transporting ATPase alpha chain 2
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit beta-1
  • Ligand: POTASSIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CHOLESTEROL
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: non gastric H,K-ATPase

SupramoleculeName: non gastric H,K-ATPase / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 150 kDa/nm

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Macromolecule #1: Potassium-transporting ATPase alpha chain 2

MacromoleculeName: Potassium-transporting ATPase alpha chain 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: H+/K+-exchanging ATPase
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 109.145664 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GMDLDDHRLS NTELEQKYGT NIIQGLSSVR ATELLARDGP NTLTPPKQTP EIIKFLKQMV GGFSILLWIG AALCWIAFVI QYVNNSASL DNVYLGAILV LVVILTGIFA YYQEAKSTNI MASFSKMIPQ QALVIRDAEK KVISAEQLVV GDVVEIKGGD Q IPADIRLV ...String:
GMDLDDHRLS NTELEQKYGT NIIQGLSSVR ATELLARDGP NTLTPPKQTP EIIKFLKQMV GGFSILLWIG AALCWIAFVI QYVNNSASL DNVYLGAILV LVVILTGIFA YYQEAKSTNI MASFSKMIPQ QALVIRDAEK KVISAEQLVV GDVVEIKGGD Q IPADIRLV FSQGCKVDNS SLTGESEPQA RSTEFTHENP LETKNIGFYS TTCLEGTATG IVINTGDRTI IGRIASLASG VG SEKTPIA IEIEHFVHIV AGVAVSIDII FFITAVCMKY YVLDAIIFLI SIIVANVPEG LLATVTVTLS LTAKRMAKKN CLV KNLEAV ETLGSTSIIC SDKTGTLTQN RMTVAHLWFD NQIFVADTSE NQTKQAFDQS SGTWASLSKI ITLCNRAEFR PGQE SVPIM KRTVVGDASE TALLKFSEVI LGDVMGIRKR NHKVAEIPFN STNKFQLSIH ETEDPNNKRF LVVMKGAPER ILEKC STIM INGQEQPLDK SSADSFHTAY MELGGLGERV LGFCHLYLPA EQFPQSYIFD VDSVNFPTSN FCFVGLLSMI DPPRST VPD AVSKCRSAGI KVIMVTGDHP ITAKAIAKSV GIISANNETV EDIAKRRNIA VEQVNKREAK AAVVTGMELK DMTPEQL DE LLTNYQEIVF ARTSPQQKLI IVEGCQRQDA IVAVTGDGVN DSPALKKADI GIAMGIAGSD AAKNAADMVL LDDNFASI V TGVEEGRLIF DNLKKTIAYT LTSNIPELCP FLIYIVAGLP LPIGTITILF IDLGTDIIPS IALAYEKAES DIMNRKPRH KKKDRLVNTQ LAIYSYLHIG LMQALGGFLV YFTVYAQQGF WPTSLINLRV AWETDDINDL EDSYGQEWTR YQRKYLEWTG STAFFVAIM IQQWADLIIC KTRRNSIFQQ GLFRNKVIWV GIASQVIVAL ILSYGLGSVP ALSFTMLRVQ YWFVAVPHAI L IWVYDEMR KLFIRLYPGS WWDKNMYY

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Macromolecule #2: Sodium/potassium-transporting ATPase subunit beta-1

MacromoleculeName: Sodium/potassium-transporting ATPase subunit beta-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 37.516859 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGDYKDDDDK SSGENLYFQG MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISELKPTYQ DRVAPPGLTQ IPQIQKTEIS FRPNDPKSYE AYVLNIIRFL EKYKDSAQKD DMIFEDCGSM PSEPKERGEF N HERGERKV ...String:
MGDYKDDDDK SSGENLYFQG MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISELKPTYQ DRVAPPGLTQ IPQIQKTEIS FRPNDPKSYE AYVLNIIRFL EKYKDSAQKD DMIFEDCGSM PSEPKERGEF N HERGERKV CRFKLDWLGN CSGLNDESYG YKEGKPCIII KLNRVLGFKP KPPKNESLET YPLTMKYNPN VLPVQCTGKR DE DKDKVGN IEYFGMGGFY GFPLQYYPYY GKLLQPKYLQ PLLAVQFTNL TLDTEIRIEC KAYGENIGYS EKDRFQGRFD VKI EVKS

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 3 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #4: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 5 / 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 #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 769755
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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