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Yorodumi- PDB-7zrl: Cryo-EM map of the unphosphorylated KdpFABC complex in the E2-P c... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7zrl | ||||||||||||||||||
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Title | Cryo-EM map of the unphosphorylated KdpFABC complex in the E2-P conformation, under turnover conditions | ||||||||||||||||||
Components |
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Keywords | MEMBRANE PROTEIN / P-type ATPase / superfamily of K+ transporters (SKT) / potassium uptake system | ||||||||||||||||||
Function / homology | Function and homology information P-type K+ transporter / P-type potassium transmembrane transporter activity / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity ...P-type K+ transporter / P-type potassium transmembrane transporter activity / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane Similarity search - Function | ||||||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å | ||||||||||||||||||
Authors | Hielkema, L. / Stock, C. / Silberberg, J.M. / Corey, R.A. / Wunnicke, D. / Dubach, V.R.A. / Stansfeld, P.J. / Haenelt, I. / Paulino, C. | ||||||||||||||||||
Funding support | United Kingdom, 5items
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Citation | Journal: Elife / Year: 2022 Title: Inhibited KdpFABC transitions into an E1 off-cycle state. Authors: Jakob M Silberberg / Charlott Stock / Lisa Hielkema / Robin A Corey / Jan Rheinberger / Dorith Wunnicke / Victor R A Dubach / Phillip J Stansfeld / Inga Hänelt / Cristina Paulino / Abstract: KdpFABC is a high-affinity prokaryotic K uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K levels, KdpFABC needs to be ...KdpFABC is a high-affinity prokaryotic K uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K levels, KdpFABC needs to be inhibited to prevent excessive K accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES motif in the A domain of the pump subunit KdpB (KdpB). Here, we explore the structural basis of inhibition by KdpB phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpB phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states. | ||||||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7zrl.cif.gz | 244 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7zrl.ent.gz | 196.5 KB | Display | PDB format |
PDBx/mmJSON format | 7zrl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7zrl_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7zrl_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7zrl_validation.xml.gz | 49.9 KB | Display | |
Data in CIF | 7zrl_validation.cif.gz | 76.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/zr/7zrl ftp://data.pdbj.org/pub/pdb/validation_reports/zr/7zrl | HTTPS FTP |
-Related structure data
Related structure data | 14918MC 7zrdC 7zreC 7zrgC 7zrhC 7zriC 7zrjC 7zrkC 7zrmC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 59218.613 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpA, b0698, JW0686 / Production host: Escherichia coli (E. coli) / Variant (production host): LB2003 / References: UniProt: P03959 |
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#2: Protein | Mass: 72331.844 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: kdpB, b0697, JW0685 / Production host: Escherichia coli (E. coli) / Variant (production host): LB2003 / References: UniProt: P03960, P-type K+ transporter |
#3: Protein | Mass: 20281.035 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: kdpC, b0696, JW0684 / Production host: Escherichia coli (E. coli) / Variant (production host): LB2003 / References: UniProt: P03961 |
#4: Protein/peptide | Mass: 2853.463 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: kdpF, b4513, JW0687 / Production host: Escherichia coli (E. coli) / Variant (production host): LB2003 / References: UniProt: P36937 |
#5: Chemical | ChemComp-K / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: KdpFABC / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
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Molecular weight | Value: 0.157 MDa / Experimental value: NO |
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: LB2003 |
Buffer solution | pH: 8 Details: 10 mM Tris-HCl pH 8, 10 mM MgCl2, 10 mM NaCl and 0.0125% DDM |
Specimen | Conc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 49407 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K |
Image recording | Average exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 11428 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60 |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 287232 | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47004 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6HRB | ||||||||||||||||||||||||||||||||
Refine LS restraints |
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