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- PDB-4ux1: Cryo-EM structure of antagonist-bound E2P gastric H,K-ATPase (SCH... -

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Basic information

Entry
Database: PDB / ID: 4ux1
TitleCryo-EM structure of antagonist-bound E2P gastric H,K-ATPase (SCH.E2. AlF)
Components
  • POTASSIUM-TRANSPORTING ATPASE ALPHA CHAIN 1
  • POTASSIUM-TRANSPORTING ATPASE SUBUNIT BETA
KeywordsTRANSPORT PROTEIN / POTASSIUM-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 / 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. / 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, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 17, 2014Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2014Group: Database references
Revision 1.2Oct 1, 2014Group: Database references
Revision 1.3Nov 12, 2014Group: Database references

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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 software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)140.900, 111.300, 320.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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


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


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 267
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: antagonist-bound E2P gastric H,K-ATPase / Type: COMPLEX
Buffer solutionpH: 4.8
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: Oct 29, 2013
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: 2997 nm / Nominal defocus min: 808 nm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM
DiffractionMean temperature: 4 K
DetectorDate: Oct 29, 2013
Radiation wavelengthRelative weight: 1
ReflectionResolution: 8→129 Å / Num. obs: 35798 / % possible obs: 52.6 % / Biso Wilson estimate: 32.4 Å2

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Processing

Software
NameVersionClassification
MRCmodel building
CNS1.3refinement
MRCSUITEdata scaling
MRCphasing
3D reconstructionResolution: 8 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 2D CRYSTAL
RefinementResolution: 8→128.95 Å / Rfactor Rfree error: 0.043 / Data cutoff high absF: 876135.35 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
Details: BULK SOLVENT MODEL USED SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2759. (DEPOSITION ID: 12786).
RfactorNum. reflection% reflectionSelection details
Rfree0.516 146 4.9 %RANDOM
Rwork0.507 ---
obs0.507 2994 52.6 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 4098.69 Å2 / ksol: 0.1 e/Å3
Displacement parametersBiso mean: 0 Å2
Baniso -1Baniso -2Baniso -3
1-25.76 Å20 Å20 Å2
2--37.87 Å20 Å2
3----63.64 Å2
Refine analyze
FreeObs
Luzzati coordinate error1.4 Å2.23 Å
Luzzati d res low-10 Å
Luzzati sigma a0.2 Å-2.74 Å
Refinement stepCycle: LAST / Resolution: 8→128.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9161 0 0 0 9161
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
ELECTRON CRYSTALLOGRAPHYc_bond_d0.024
ELECTRON CRYSTALLOGRAPHYc_bond_d_na
ELECTRON CRYSTALLOGRAPHYc_bond_d_prot
ELECTRON CRYSTALLOGRAPHYc_angle_d
ELECTRON CRYSTALLOGRAPHYc_angle_d_na
ELECTRON CRYSTALLOGRAPHYc_angle_d_prot
ELECTRON CRYSTALLOGRAPHYc_angle_deg2.8
ELECTRON CRYSTALLOGRAPHYc_angle_deg_na
ELECTRON CRYSTALLOGRAPHYc_angle_deg_prot
ELECTRON CRYSTALLOGRAPHYc_dihedral_angle_d25.1
ELECTRON CRYSTALLOGRAPHYc_dihedral_angle_d_na
ELECTRON CRYSTALLOGRAPHYc_dihedral_angle_d_prot
ELECTRON CRYSTALLOGRAPHYc_improper_angle_d5.51
ELECTRON CRYSTALLOGRAPHYc_improper_angle_d_na
ELECTRON CRYSTALLOGRAPHYc_improper_angle_d_prot
ELECTRON CRYSTALLOGRAPHYc_mcbond_it01.5
ELECTRON CRYSTALLOGRAPHYc_mcangle_it02
ELECTRON CRYSTALLOGRAPHYc_scbond_it02
ELECTRON CRYSTALLOGRAPHYc_scangle_it02.5
Refine LS restraints NCSNCS model details: NONE
LS refinement shellResolution: 8→8.5 Å / Rfactor Rfree error: 0.224 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.633 8 3 %
Rwork0.51 262 -
obs--29.4 %
Xplor fileSerial no: 1 / Param file: PROTEIN_REP.PARAM / Topol file: PROTEIN.TOP

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