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- EMDB-2759: Cryo-EM structure of antagonist-bound E2P gastric H+,K+-ATPase (S... -

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Basic information

Entry
Database: EMDB / ID: EMD-2759
TitleCryo-EM structure of antagonist-bound E2P gastric H+,K+-ATPase (SCH.E2.AlF)
Map dataReconstruction of H+,K+-ATPase
Sample
  • Sample: POTASSIUM-TRANSPORTING ATPASE
  • Protein or peptide: H+,K+-ATPase
KeywordsPOTASSIUM-TRANSPORTING ATPASE
Function / homology
Function and homology information


H+/K+-exchanging ATPase / potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / P-type sodium:potassium-exchanging transporter activity / ATP biosynthetic process / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis ...H+/K+-exchanging ATPase / potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / P-type sodium:potassium-exchanging transporter activity / ATP biosynthetic process / 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 binding / potassium ion transmembrane transport / proton transmembrane transport / cell adhesion / apical plasma membrane / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane
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 / 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, 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
Potassium-transporting ATPase subunit beta / Potassium-transporting ATPase alpha chain 1
Similarity search - Component
Biological speciesSus scrofa (pig)
Methodelectron crystallography / cryo EM / Resolution: 8.0 Å
AuthorsAbe K / Tani K / Fujiyoshi Y
CitationJournal: J Biol Chem / Year: 2014
Title: Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.
Authors: Kazuhiro Abe / Kazutoshi Tani / Yoshinori Fujiyoshi /
Abstract: Gastric H(+),K(+)-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an ...Gastric H(+),K(+)-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K(+)-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H(+),K(+)-ATPase is consistent with that observed in sarcoplasmic reticulum Ca(2+)-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.
History
DepositionAug 18, 2014-
Header (metadata) releaseSep 10, 2014-
Map releaseSep 17, 2014-
UpdateNov 12, 2014-
Current statusNov 12, 2014Processing site: PDBe / Status: Released

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Structure visualization

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  • Surface view with section colored by density value
  • Surface level: 1
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  • Surface view colored by height
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  • Surface view with fitted model
  • Atomic models: PDB-4ux1
  • Surface level: 1
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  • Surface view with fitted model
  • Atomic models: PDB-4ux1
  • Surface level: 1
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4ux1
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Structure viewerEM map:
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Supplemental images

EMD-2759-HU_...

Downloads & links

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Map

FileDownload / File: emd_2759.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of H+,K+-ATPase
Voxel sizeX: 1.96 Å / Y: 1.86 Å / Z: 2 Å
Density
Contour LevelBy EMDB: 1.6 / Movie #1: 1
Minimum - Maximum-4.35360003 - 6.19469976
Average (Standard dev.)-0.00324994 (±0.99307626)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-36-30-80
Dimensions7361161
Spacing6173161
CellA: 143.08 Å / B: 113.46 Å / C: 322.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.961.862
M x/y/z7361161
origin x/y/z0.0000.0000.000
length x/y/z143.080113.460322.000
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-4.3546.195-0.003

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Supplemental data

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Segmentation: This mask represent a diprotomer of HK-ATPase in 2D crystal.

AnnotationThis mask represent a diprotomer of HK-ATPase in 2D crystal.
Fileemd_2759_msk_1.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 : POTASSIUM-TRANSPORTING ATPASE

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

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Supramolecule #1000: POTASSIUM-TRANSPORTING ATPASE

SupramoleculeName: POTASSIUM-TRANSPORTING ATPASE / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: H+,K+-ATPase

MacromoleculeName: H+,K+-ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: One alpha and one beta chain of HK-ATPase / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: gastric / Location in cell: Plasma membrane
Molecular weightTheoretical: 110 KDa
SequenceUniProtKB: Potassium-transporting ATPase alpha chain 1 / GO: 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: 20 mM propionate, 1 mM MgCl2, 1 mM AlCl3, 4 mM NaF, 1 mM ADP, 3 mM DTT and 10 M SCH28080 at pH 4.8 with Tris.
GridDetails: molybdenum grid with thin carbon support
VitrificationCryogen name: NITROGEN / Instrument: LEICA KF80
Details: Vitrification carried out in cold room at 4 degrees Celsius
DetailsCrystals grown in dialysis
Crystal formationDetails: Crystals grown in dialysis

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 2.997 µm / Nominal defocus min: 0.808 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Helium cooled / Specimen holder model: JEOL / Tilt angle min: -63.6 / Tilt angle max: 63.6 / Tilt series - Axis1 - Min angle: -63.6 ° / Tilt series - Axis1 - Max angle: 63.6 °
TemperatureMin: 4 K / Average: 4 K
DateOct 29, 2013
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 267 / Average electron dose: 20 e/Å2 / Bits/pixel: 14

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Image processing

Crystal parametersUnit cell - A: 140.9 Å / Unit cell - B: 111.3 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 micrographs
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: MRC
DetailsImages were processed using MRC suite.

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