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- PDB-2yn9: Cryo-EM structure of gastric H+,K+-ATPase with bound rubidium -

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Basic information

Entry
Database: PDB / ID: 2yn9
TitleCryo-EM structure of gastric H+,K+-ATPase with bound rubidium
Components
  • POTASSIUM-TRANSPORTING ATPASE ALPHA CHAIN 1
  • POTASSIUM-TRANSPORTING ATPASE SUBUNIT BETA
KeywordsHYDROLASE / P-TYPE ATPASE / PROTON PUMP
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 / 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 ...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 / 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 / haloacid dehalogenase-like hydrolase / Cation-transporting P-type ATPase, C-terminal ...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 / haloacid dehalogenase-like hydrolase / 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
Potassium-transporting ATPase subunit beta / Potassium-transporting ATPase alpha chain 1
Similarity search - Component
Biological speciesSUS SCROFA (pig)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 8 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 13, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 7, 2012Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2013Group: Database references / Derived calculations / Other
Revision 1.2Jul 16, 2014Group: Other

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Structure visualization

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Assembly

Deposited unit
A: POTASSIUM-TRANSPORTING ATPASE ALPHA CHAIN 1
B: POTASSIUM-TRANSPORTING ATPASE SUBUNIT BETA


Theoretical massNumber of molelcules
Total (without water)147,5142
Polymers147,5142
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)141.000, 110.600, 320.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121

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Components

#1: Protein POTASSIUM-TRANSPORTING ATPASE ALPHA CHAIN 1 / GASTRIC H(+)/K(+) ATPASE SUBUNIT ALPHA / PROTON PUMP


Mass: 114399.688 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SUS SCROFA (pig) / Organ: STOMACH / References: UniProt: P19156, H+/K+-exchanging ATPase
#2: Protein POTASSIUM-TRANSPORTING ATPASE SUBUNIT BETA / GASTRIC H(+)/K(+) ATPASE SUBUNIT BETA / PROTON PUMP BETA CHAIN / GP60-90


Mass: 33113.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) SUS SCROFA (pig) / Organ: STOMACH / References: UniProt: P18434

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 248
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: gastric H+,K+-ATPase with bound rubidium / Type: COMPLEX
Buffer solutionpH: 4.8
Details: 20mM propionate, 1mM ADP, 3mM DTT,pH 4.8-4.9 adjusted by Tris, 1mM MgCl2, 1mM AlCl3, 4mM NaF, 10mM RbCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA KF80 / Cryogen name: NITROGEN
Crystal growpH: 4.8 / Details: pH 4.8

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Data collection

MicroscopyModel: JEOL KYOTO-3000SFF / Date: Mar 23, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 40000 X / Nominal defocus max: 3480 nm / Nominal defocus min: 830 nm
Image recordingFilm or detector model: KODAK SO-163 FILM
DiffractionMean temperature: 4 K
DetectorDate: Mar 23, 2010
Radiation wavelengthRelative weight: 1
ReflectionResolution: 8→129 Å / Num. obs: 39197 / % possible obs: 73.2 %

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Processing

Software
NameVersionClassification
MRCmodel building
SITUSrefinement
MRCSUITEdata scaling
MRCphasing
3D reconstructionResolution: 8 Å / Symmetry type: 2D CRYSTAL
RefinementResolution: 8→129 Å / Num. reflection obs: 4166 / σ(F): 0
Details: ALL REGIONS WERE MODELED STEREOCHEMICALLY. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2219. (DEPOSITION ID: 11197).
Refinement stepCycle: LAST / Resolution: 8→129 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9694 0 0 0 9694
Refine LS restraints
Refine-IDTypeDev ideal
ELECTRON CRYSTALLOGRAPHYo_bond_d0.018
ELECTRON CRYSTALLOGRAPHYo_bond_d_na
ELECTRON CRYSTALLOGRAPHYo_bond_d_prot
ELECTRON CRYSTALLOGRAPHYo_angle_d
ELECTRON CRYSTALLOGRAPHYo_angle_d_na
ELECTRON CRYSTALLOGRAPHYo_angle_d_prot
ELECTRON CRYSTALLOGRAPHYo_angle_deg2.1
ELECTRON CRYSTALLOGRAPHYo_angle_deg_na
ELECTRON CRYSTALLOGRAPHYo_angle_deg_prot
ELECTRON CRYSTALLOGRAPHYo_dihedral_angle_d
ELECTRON CRYSTALLOGRAPHYo_dihedral_angle_d_na
ELECTRON CRYSTALLOGRAPHYo_dihedral_angle_d_prot
ELECTRON CRYSTALLOGRAPHYo_improper_angle_d
ELECTRON CRYSTALLOGRAPHYo_improper_angle_d_na
ELECTRON CRYSTALLOGRAPHYo_improper_angle_d_prot
ELECTRON CRYSTALLOGRAPHYo_mcbond_it
ELECTRON CRYSTALLOGRAPHYo_mcangle_it
ELECTRON CRYSTALLOGRAPHYo_scbond_it
ELECTRON CRYSTALLOGRAPHYo_scangle_it

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