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- PDB-6hra: Cryo-EM structure of the KdpFABC complex in an E1 outward-facing ... -

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Basic information

Entry
Database: PDB / ID: 6hra
TitleCryo-EM structure of the KdpFABC complex in an E1 outward-facing state (state 1)
Components
  • Potassium-transporting ATPase ATP-binding subunit
  • Potassium-transporting ATPase KdpC subunit
  • Potassium-transporting ATPase KdpF subunit
  • Potassium-transporting ATPase potassium-binding subunit
KeywordsMEMBRANE PROTEIN / P-type ATPase superfamily of K+ transporters (SKT) potassium uptake system four subunit complex
Function / homology
Function and homology information


P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity ...P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / 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
Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / 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 potassium-binding subunit / Potassium-transporting ATPase ATP-binding subunit / Potassium-transporting ATPase KdpC subunit / Potassium-transporting ATPase KdpF subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsStock, C. / Hielkema, L. / Tascon, I. / Wunnicke, D. / Oostergetel, G.T. / Azkargorta, M. / Paulino, C. / Haenelt, I.
Funding support Germany, Netherlands, 3items
OrganizationGrant numberCountry
German Research FoundationHA 6322/3-1 Germany
Netherlands Organisation for Scientific Research722.017.001 Netherlands
European Research Council749732 Netherlands
CitationJournal: Nat Commun / Year: 2018
Title: Cryo-EM structures of KdpFABC suggest a K transport mechanism via two inter-subunit half-channels.
Authors: C Stock / L Hielkema / I Tascón / D Wunnicke / G T Oostergetel / M Azkargorta / C Paulino / I Hänelt /
Abstract: P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K uptake system KdpFABC is unique. While ATP hydrolysis is accomplished ...P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K uptake system KdpFABC is unique. While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. Here, we report two cryo-EM structures of the 157 kDa, asymmetric KdpFABC complex at 3.7 Å and 4.0 Å resolution in an E1 and an E2 state, respectively. Unexpectedly, the structures suggest a translocation pathway through two half-channels along KdpA and KdpB, uniting the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K channels. This way, KdpFABC would function as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms.
History
DepositionSep 26, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 5, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Potassium-transporting ATPase potassium-binding subunit
C: Potassium-transporting ATPase KdpC subunit
D: Potassium-transporting ATPase KdpF subunit
B: Potassium-transporting ATPase ATP-binding subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,0377
Polymers154,9194
Non-polymers1173
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15750 Å2
ΔGint-139 kcal/mol
Surface area53660 Å2
MethodPISA

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Components

#1: Protein Potassium-transporting ATPase potassium-binding subunit / ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit A / Potassium-translocating ATPase A chain


Mass: 59218.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: kdpA, b0698, JW0686 / Production host: Escherichia coli (E. coli) / References: UniProt: P03959
#2: Protein Potassium-transporting ATPase KdpC subunit / ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit C / Potassium-translocating ATPase C chain


Mass: 20281.035 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: kdpC, b0696, JW0684 / Production host: Escherichia coli (E. coli) / References: UniProt: P03961
#3: Protein/peptide Potassium-transporting ATPase KdpF subunit / ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit F / Potassium-translocating ATPase F chain


Mass: 3071.714 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: kdpF, b4513, JW0687 / Production host: Escherichia coli (E. coli) / References: UniProt: P36937
#4: Protein Potassium-transporting ATPase ATP-binding subunit / ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit B / Potassium-translocating ATPase B chain


Mass: 72347.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: kdpB, b0697, JW0685 / Production host: Escherichia coli (E. coli) / References: UniProt: P03960, EC: 3.6.3.12
#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: KdpFABC / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.157 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 10 mM Tris-HCl pH 8, 10 mM MgCl2, 10 mM NaCl and 0.012% DDM
SpecimenConc.: 3.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49407 X / Calibrated magnification: 49407 X / Nominal defocus max: 3000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage 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: 9 / Num. of real images: 7327
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
2EPU1.9.1image acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2.1binitial Euler assignment
11RELION2.1bfinal Euler assignment
13RELION2.1b3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 826403
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219897 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5MRW

5mrw

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00711015
ELECTRON MICROSCOPYf_angle_d0.89914993
ELECTRON MICROSCOPYf_dihedral_angle_d6.7216554
ELECTRON MICROSCOPYf_chiral_restr0.0541809
ELECTRON MICROSCOPYf_plane_restr0.0071902

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