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- EMDB-2220: Cryo-EM structure of gastric H+,K+-ATPase with bound K+ -

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Basic information

Entry
Database: EMDB / ID: EMD-2220
TitleCryo-EM structure of gastric H+,K+-ATPase with bound K+
Map dataReconstruction of H,K-ATPase with bound K
Sample
  • Sample: Pig Gastric H,K-ATPase
  • Protein or peptide: POTASSIUM-TRANSPORTING ATPASE
KeywordsP-type ATPase / proton pump
Function / homologyATP biosynthetic process / P-type ATPase, A domain superfamily
Function and homology information
Biological speciesSus scrofa (pig)
Methodelectron crystallography / cryo EM / Resolution: 8.0 Å
AuthorsAbe K / Tani K / Friedrich T / Fujiyoshi Y
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site.
Authors: Kazuhiro Abe / Kazutoshi Tani / Thomas Friedrich / Yoshinori Fujiyoshi /
Abstract: Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a ...Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen.
History
DepositionOct 12, 2012-
Header (metadata) releaseNov 14, 2012-
Map releaseNov 14, 2012-
UpdateNov 14, 2012-
Current statusNov 14, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2220.jpg

Downloads & links

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Map

FileDownload / File: emd_2220.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of H,K-ATPase with bound K
Voxel sizeX: 1.8433 Å / Y: 1.9583 Å / Z: 2 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-6.25960016 - 6.88040018
Average (Standard dev.)-0.00338815 (±0.99191403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-36-30-80
Dimensions7361161
Spacing6173161
CellA: 134.5609 Å / B: 119.4563 Å / C: 322.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.8433013698631.95829508196722
M x/y/z7361161
origin x/y/z0.0000.0000.000
length x/y/z134.561119.456322.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S213
start NC/NR/NS-30-36-80
NC/NR/NS6173161
D min/max/mean-6.2606.880-0.003

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Supplemental data

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Sample components

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Entire : Pig Gastric H,K-ATPase

EntireName: Pig Gastric H,K-ATPase
Components
  • Sample: Pig Gastric H,K-ATPase
  • Protein or peptide: POTASSIUM-TRANSPORTING ATPASE

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Supramolecule #1000: Pig Gastric H,K-ATPase

SupramoleculeName: Pig Gastric H,K-ATPase / type: sample / ID: 1000 / Oligomeric state: One alpha and one beta chain of HK-ATPase / Number unique components: 1
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: POTASSIUM-TRANSPORTING ATPASE

MacromoleculeName: POTASSIUM-TRANSPORTING ATPASE / type: protein_or_peptide / ID: 1 / Name.synonym: H+,K+-ATPase / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: stomach / Location in cell: Plasma membrane
Molecular weightTheoretical: 150 KDa
SequenceGO: ATP biosynthetic process / InterPro: P-type ATPase, A domain superfamily

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration8 mg/mL
BufferpH: 4.8
Details: 20mM propionate, 1mM ADP, 3mM DTT, ph4.8-4.9 adjusted by Tris, 1mM MgCl2, 1mM AlCl3, 4mM NaF, 10mM KCl
GridDetails: Molybdenum grid
VitrificationCryogen name: NITROGEN / Instrument: LEICA KF80
Details: The grids were blotted with filter paper and fast frozen into liquid nitrogen
DetailsCrystals grown by dialysis
Crystal formationDetails: Crystals grown by dialysis

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 3.88 µm / Nominal defocus min: 0.67 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Helium cooled / Specimen holder model: JEOL / Tilt angle min: -60 / Tilt angle max: 60 / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
DateFeb 15, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 278 / Bits/pixel: 12

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Image processing

Crystal parametersUnit cell - A: 141.0 Å / Unit cell - B: 110.6 Å / Unit cell - C: 320.0 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 2 21 21
CTF correctionDetails: Each image
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Software - Name: MRC
DetailsImages were processed using MRC suite.

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