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- EMDB-31294: Cryo-EM structure of the gastric proton pump K791S/E820D/Y340N/E9... -

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Basic information

Entry
Database: EMDB / ID: EMD-31294
TitleCryo-EM structure of the gastric proton pump K791S/E820D/Y340N/E936V/Y799W mutant in K+-occluded (K+)E2-AlF state
Map data
Sample
  • Complex: alpha-beta complex of the gastric proton pump
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: Potassium-transporting ATPase subunit beta
  • Ligand: MAGNESIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: POTASSIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: CHOLESTEROL
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
Function / homology
Function and homology information


potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / potassium ion transmembrane transport ...potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / potassium ion transmembrane transport / proton transmembrane transport / cell adhesion / apical plasma membrane / magnesium ion binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / 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 ...Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / 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 / 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 / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Sodium/potassium-transporting ATPase subunit alpha / Potassium-transporting ATPase subunit beta
Similarity search - Component
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsAbe K
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Nat Commun / Year: 2021
Title: Gastric proton pump with two occluded K engineered with sodium pump-mimetic mutations.
Authors: Kazuhiro Abe / Kenta Yamamoto / Katsumasa Irie / Tomohiro Nishizawa / Atsunori Oshima /
Abstract: The gastric H,K-ATPase mediates electroneutral exchange of 1H/1K per ATP hydrolysed across the membrane. Previous structural analysis of the K-occluded E2-P transition state of H,K-ATPase showed a ...The gastric H,K-ATPase mediates electroneutral exchange of 1H/1K per ATP hydrolysed across the membrane. Previous structural analysis of the K-occluded E2-P transition state of H,K-ATPase showed a single bound K at cation-binding site II, in marked contrast to the two K ions occluded at sites I and II of the closely-related Na,K-ATPase which mediates electrogenic 3Na/2K translocation across the membrane. The molecular basis of the different K stoichiometry between these K-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H,K-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K bound H,K-ATPase, achieved by five mutations, including amino acids directly coordinating K (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K-occluded conformation (Tyr799Trp). This quintuple mutant in the K-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K accommodated at their cation-binding site.
History
DepositionMay 12, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7et1
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31294.png

Downloads & links

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Map

FileDownload / File: emd_31294.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.011 / Movie #1: 0.011
Minimum - Maximum-0.028202144 - 0.069183744
Average (Standard dev.)0.00014878769 (±0.0024419227)
SymmetrySpace group: 1
Details

EMDB XML:

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

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.480212.480212.480
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0280.0690.000

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Supplemental data

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Mask #1

Fileemd_31294_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_31294_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_31294_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : alpha-beta complex of the gastric proton pump

EntireName: alpha-beta complex of the gastric proton pump
Components
  • Complex: alpha-beta complex of the gastric proton pump
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha
    • Protein or peptide: Potassium-transporting ATPase subunit beta
  • Ligand: MAGNESIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: POTASSIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: CHOLESTEROL
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: alpha-beta complex of the gastric proton pump

SupramoleculeName: alpha-beta complex of the gastric proton pump / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 135 KDa

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

MacromoleculeName: Sodium/potassium-transporting ATPase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 109.099547 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AMEINDHQLS VAELEQKYQT SATKGLSASL AAELLLRDGP NALRPPRGTP EYVKFARQLA GGLQCLMWVA AAICLIAFAI QASEGDLTT DDNLYLALAL IAVVVVTGCF GYYQEFKSTN IIASFKNLVP QQATVIRDGD KFQINADQLV VGDLVEMKGG D RVPADIRI ...String:
AMEINDHQLS VAELEQKYQT SATKGLSASL AAELLLRDGP NALRPPRGTP EYVKFARQLA GGLQCLMWVA AAICLIAFAI QASEGDLTT DDNLYLALAL IAVVVVTGCF GYYQEFKSTN IIASFKNLVP QQATVIRDGD KFQINADQLV VGDLVEMKGG D RVPADIRI LQAQGCKVDN SSLTGESEPQ TRSPECTHES PLETRNIAFF STMCLEGTAQ GLVVNTGDRT IIGRIASLAS GV ENEKTPI AIEIEHFVDI IAGLAILFGA TFFIVAMCIG YTFLRAMVFF MAIVVANVPE GLLATVTVCL SLTAKRLASK NCV VKNLEA VETLGSTSVI CSDKTGTLTQ NRMTVSHLWF DNHIHSADTT EDQSGQTFDQ SSETWRALCR VLTLCNRAAF KSGQ DAVPV PKRIVIGDAS ETALLKFSEL TLGNAMGYRE RFPKVCEIPF NSTNKFQLSI HTLEDPRDPR HVLVMKGAPE RVLER CSSI LIKGQELPLD EQWREAFQTA YLSLGGLGER VLGFCQLYLS EKDYPPGYAF DVEAMNFPTS GLCFAGLVSM IDPPRA TVP DAVLKCRTAG IRVIMVTGDH PITAKAIAAS VGIISEGSET VEDIAARLRV PVDQVNRKDA RACVINGMQL KDMDPSE LV EALRTHPEMV FARTSPQQKL VIVESCQRLG AIVAVTGDGV NDSPALKKAD IGVAMGIAGS DAAKNAADMI LLDDNFAS I VTGVEQGRLI FDNLKKSIAY TLTSNIPELT PWLIYITVSV PLPLGCITIL FIDLCTDIFP SVSLAYEKAE SDIMHLRPR NPKRDRLVNE PLAAYSYFQI GAIQSFAGFT DYFTAMAQEG WFPLLCVGLR PQWENHHLQD LQDSYGQEWT FGQRLYQQYT CYTVFFISI VMCQIADVLI RKTRRLSAFQ QGFFRNRILV IAIVFQVCIG CFLCYCPGMP NIFNFMPIRF QWWLVPMPFS L LIFVYDEI RKLGVRCCPG SWWDQELYY

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Macromolecule #2: Potassium-transporting ATPase subunit beta

MacromoleculeName: Potassium-transporting ATPase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 29.762107 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLGRTLSRWV WISLYYVAFY VVMSGIFALC IYVLMRTIDP YTPDYQDQLK SPGVTLRPDV YGEKGLDISY NVSDSTTWAG LAHTLHRFL AGYSPAAQEG SINCTSEKYF FQESFLAPNH TKFSCKFTAD MLQNCSGRPD PTFGFAEGKP CFIIKMNRIV K FLPGNSTA ...String:
MLGRTLSRWV WISLYYVAFY VVMSGIFALC IYVLMRTIDP YTPDYQDQLK SPGVTLRPDV YGEKGLDISY NVSDSTTWAG LAHTLHRFL AGYSPAAQEG SINCTSEKYF FQESFLAPNH TKFSCKFTAD MLQNCSGRPD PTFGFAEGKP CFIIKMNRIV K FLPGNSTA PRVDCAFLDQ PRDGPPLQVE YFPANGTYSL HYFPYYGKKA QPHYSNPLVA AKLLNVPRNR DVVIVCKILA EH VSFDNPH DPYEGKVEFK LKIQK

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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: 3 / 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: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 7 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

BufferpH: 6.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300
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: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 72308
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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