EMDB-2219: Cryo-EM structure of gastric H+,K+-ATPase with bound Rb+

Basic information

• Entry: Database: EMDB / ID: EMD-2219
• Title: Cryo-EM structure of gastric H+,K+-ATPase with bound Rb+
• Map data: Reconstruction of H+,K+-ATPase with Rb+

Sample

• Sample: H+,K+-ATPase bound with Rb+
• Protein or peptide: POTASSIUM-TRANSPORTING ATPASE

• Keywords: P-type ATPase / Gastric H+/K+-ATPase / proton pump

Function / homology

Function:

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

Domain/homology:

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

Component:

Potassium-transporting ATPase subunit beta / Potassium-transporting ATPase alpha chain 1

• Biological species: Sus scrofa (pig)
• Method: electron crystallography / cryo EM / Resolution: 8.0 Å
• Authors: Abe K / Tani K / Friedrich T / Fujiyoshi Y
• Citation: Journal: 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 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

Deposition	Oct 12, 2012	-
Header (metadata) release	Nov 14, 2012	-
Map release	Nov 14, 2012	-
Update	Nov 14, 2012	-
Current status	Nov 14, 2012	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_2219.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstruction of H+,K+-ATPase with Rb+
• Voxel size: X: 1.8433 Å / Y: 1.9583 Å / Z: 2 Å

Density

Contour Level	By AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum	-4.47049999 - 6.63910007
Average (Standard dev.)	-0.00531801 (±0.99277723)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Y X Z Origin -36 -30 -80 Dimensions 73 61 161 Spacing 61 73 161
Cell	A: 134.5609 Å / B: 119.4563 Å / C: 322.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.843301369863	1.9582950819672	2
M x/y/z	73	61	161
origin x/y/z	0.000	0.000	0.000
length x/y/z	134.561	119.456	322.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-36	-30	-80
NX/NY/NZ	73	61	161
MAP C/R/S	2	1	3
start NC/NR/NS	-30	-36	-80
NC/NR/NS	61	73	161
D min/max/mean	-4.470	6.639	-0.005

Supplemental data

Sample components

Entire : H+,K+-ATPase bound with Rb+

• Entire: Name: H+,K+-ATPase bound with Rb+

Components

• Sample: H+,K+-ATPase bound with Rb+
• Protein or peptide: POTASSIUM-TRANSPORTING ATPASE

Supramolecule #1000: H+,K+-ATPase bound with Rb+

• Supramolecule: Name: H+,K+-ATPase bound with Rb+ / type: sample / ID: 1000 / Oligomeric state: One alpha and one beta chain of HK-ATPase / Number unique components: 1
• Molecular weight: Theoretical: 150 KDa

Macromolecule #1: POTASSIUM-TRANSPORTING ATPASE

• Macromolecule: Name: POTASSIUM-TRANSPORTING ATPASE / type: protein_or_peptide / ID: 1 / 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 weight: Theoretical: 150 KDa
• Sequence: GO: ATP biosynthetic process / InterPro: P-type ATPase, A domain superfamily

Experimental details

Structure determination

• Method: cryo EM
• Processing: electron crystallography
• Aggregation state: 2D array

Sample preparation

• Concentration: 8 mg/mL
• Buffer: pH: 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
• Grid: Details: molybdenum grid with thin carbon support
• Vitrification: Cryogen name: NITROGEN / Instrument: LEICA KF80; Details: Vitrification carried out in cold room at 4 degrees Celsius
• Details: Crystals grown in dialysis
• Crystal formation: Details: Crystals grown in dialysis

Electron microscopy

• Microscope: JEOL KYOTO-3000SFF
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 39500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 3.48 µm / Nominal defocus min: 0.83 µm / Nominal magnification: 40000
• Sample stage: Specimen 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 °
• Date: Mar 23, 2010
• Image recording: Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 248 / Bits/pixel: 12

Image processing

• Crystal parameters: Unit 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 correction: Details: Each image
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Software - Name: MRC
• Details: Images were processed using MRC suite.