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- PDB-8k1l: Cryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8k1l | ||||||
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Title | Cryo-EM structure of Na+,K+-ATPase alpha2 from Artemia salina in cation-free E2P form | ||||||
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![]() | TRANSPORT PROTEIN / P-type ATPase / sodium pump / membrane protein / transporter | ||||||
Function / homology | TETRAFLUOROALUMINATE ION![]() | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å | ||||||
![]() | Abe, K. / Artigas, P. | ||||||
Funding support | ![]()
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![]() | ![]() Title: A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities. Authors: Pablo Artigas / Dylan J Meyer / Victoria C Young / Kerri Spontarelli / Jessica Eastman / Evan Strandquist / Huan Rui / Benoît Roux / Matthew A Birk / Hanayo Nakanishi / Kazuhiro Abe / Craig Gatto / ![]() ![]() Abstract: Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange ...Brine shrimp () are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na,K-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na in exchange for 2 K per hydrolyzed ATP. express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines ( α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that express two salinity-independent canonical α subunits (α1 and α3), as well as two β variants, in addition to the salinity-controlled α2. These β subunits permitted heterologous expression of the α2 pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2 pumps and compared it to that of α1 (and its α2-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2-like characteristics to α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2's Lys308 helps to maintain high affinity for external K when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K-congener Rb, we prove that double-lysine-substituted pumps transport 2Na and 1 K per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing to maintain steeper Na gradients in hypersaline environments. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 278.3 KB | Display | ![]() |
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PDB format | ![]() | 204.5 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.3 MB | Display | ![]() |
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Full document | ![]() | 1.3 MB | Display | |
Data in XML | ![]() | 59.1 KB | Display | |
Data in CIF | ![]() | 85.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 36794MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 111144.211 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() |
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#2: Protein | Mass: 38496.422 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() |
#3: Chemical | ChemComp-ALF / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Na+,K+-ATPase alpha2/beta2 from Artemia Salina / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 0.13 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() |
Buffer solution | pH: 7 |
Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Microscopy | Model: JEOL CRYO ARM 300 |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
CTF correction | Type: NONE |
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3D reconstruction | Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39483 / Symmetry type: POINT |
Refinement | Cross valid method: NONE |