+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12186 | ||||||||||||
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Title | Cryo-EM Structure of KdpFABC in E2Pi state with BeF3 and K+ | ||||||||||||
Map data | non-uniform refinement by cryoSPARC | ||||||||||||
Sample |
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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 | single particle reconstruction / cryo EM / Resolution: 3.0 Å | ||||||||||||
Authors | Sweet ME / Larsen C / Pedersen BP / Stokes DL | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Structural basis for potassium transport in prokaryotes by KdpFABC. Authors: Marie E Sweet / Casper Larsen / Xihui Zhang / Michael Schlame / Bjørn P Pedersen / David L Stokes / Abstract: KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K ...KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K or water and which define a pathway for K transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K to the cytoplasm. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12186.map.gz | 118.1 MB | EMDB map data format | |
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Header (meta data) | emd-12186-v30.xml emd-12186.xml | 28.4 KB 28.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12186_fsc.xml | 11.2 KB | Display | FSC data file |
Images | emd_12186.png | 78.4 KB | ||
Masks | emd_12186_msk_1.map | 125 MB | Mask map | |
Others | emd_12186_half_map_1.map.gz emd_12186_half_map_2.map.gz | 116.2 MB 116.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12186 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12186 | HTTPS FTP |
-Validation report
Summary document | emd_12186_validation.pdf.gz | 506.9 KB | Display | EMDB validaton report |
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Full document | emd_12186_full_validation.pdf.gz | 506.5 KB | Display | |
Data in XML | emd_12186_validation.xml.gz | 19 KB | Display | |
Data in CIF | emd_12186_validation.cif.gz | 24.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12186 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12186 | HTTPS FTP |
-Related structure data
Related structure data | 7bh2MC 7bgyC 7bh1C 7lc3C 7lc6C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12186.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | non-uniform refinement by cryoSPARC | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.079 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_12186_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Half Map A from non-uniform refinement in cryoSPARC
File | emd_12186_half_map_1.map | ||||||||||||
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Annotation | Half Map A from non-uniform refinement in cryoSPARC | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map B from non-uniform refinement in cryoSPARC
File | emd_12186_half_map_2.map | ||||||||||||
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Annotation | Half Map B from non-uniform refinement in cryoSPARC | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : KdpFABC in E2Pi state with BeF3 and K+
+Supramolecule #1: KdpFABC in E2Pi state with BeF3 and K+
+Macromolecule #1: Potassium-transporting ATPase potassium-binding subunit
+Macromolecule #2: Potassium-transporting ATPase ATP-binding subunit
+Macromolecule #3: Potassium-transporting ATPase KdpC subunit
+Macromolecule #4: Potassium-transporting ATPase KdpF subunit
+Macromolecule #5: POTASSIUM ION
+Macromolecule #6: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)...
+Macromolecule #7: MAGNESIUM ION
+Macromolecule #8: BERYLLIUM TRIFLUORIDE ION
+Macromolecule #9: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 4.5 mg/mL | ||||||||||||||||||||||||
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Buffer | pH: 7.5 Component:
Details: The beryllium fluoride in the buffer was added to the protein mixture containing all other buffer components in the form of a pre-incubated mixture, at a 1:10 volume, to produce the final ...Details: The beryllium fluoride in the buffer was added to the protein mixture containing all other buffer components in the form of a pre-incubated mixture, at a 1:10 volume, to produce the final concentrations 2.5 mM BeSO4 and 10 mM NaF. | ||||||||||||||||||||||||
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | ||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0, no wait before plunging.. | ||||||||||||||||||||||||
Details | DM-solublized KdpFABC complex purified through SEC the same day as grid freezing. Peak fraction diluted to 4.5 mg/mL and complexed with magnesium fluoride solution for >30 min at room temperature prior to application to grid. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 77.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 2567 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |