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- EMDB-12186: Cryo-EM Structure of KdpFABC in E2Pi state with BeF3 and K+ -

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Basic information

Entry
Database: EMDB / ID: EMD-12186
TitleCryo-EM Structure of KdpFABC in E2Pi state with BeF3 and K+
Map datanon-uniform refinement by cryoSPARC
Sample
  • Complex: KdpFABC in E2Pi state with BeF3 and K+
    • Protein or peptide: Potassium-transporting ATPase potassium-binding subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit
    • Protein or peptide: Potassium-transporting ATPase KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit
  • Ligand: POTASSIUM IONPotassium
  • Ligand: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: water
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 K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSweet ME / Larsen C / Pedersen BP / Stokes DL
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM108043 United States
European Research Council (ERC)637372
Danish Council for Independent ResearchDFF-8021-00161
CitationJournal: 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
DepositionJan 9, 2021-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateFeb 9, 2022-
Current statusFeb 9, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bh2
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12186.png

Downloads & links

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Map

FileDownload / File: emd_12186.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnon-uniform refinement by cryoSPARC
Voxel sizeX=Y=Z: 1.079 Å
Density
Contour LevelBy AUTHOR: 0.14 / Movie #1: 0.17
Minimum - Maximum-0.96573263 - 1.4849855
Average (Standard dev.)0.00029139954 (±0.025805563)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0791.0791.079
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z345.280345.280345.280
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.9661.4850.000

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Supplemental data

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Mask #1

Fileemd_12186_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A from non-uniform refinement in cryoSPARC

Fileemd_12186_half_map_1.map
AnnotationHalf Map A from non-uniform refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B from non-uniform refinement in cryoSPARC

Fileemd_12186_half_map_2.map
AnnotationHalf Map B from non-uniform refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : KdpFABC in E2Pi state with BeF3 and K+

EntireName: KdpFABC in E2Pi state with BeF3 and K+
Components
  • Complex: KdpFABC in E2Pi state with BeF3 and K+
    • Protein or peptide: Potassium-transporting ATPase potassium-binding subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit
    • Protein or peptide: Potassium-transporting ATPase KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit
  • Ligand: POTASSIUM IONPotassium
  • Ligand: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: water

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Supramolecule #1: KdpFABC in E2Pi state with BeF3 and K+

SupramoleculeName: KdpFABC in E2Pi state with BeF3 and K+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: KdpFABC complex solublized in decyl maltopyranoside (DM) detergent.
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: TK2498 / Recombinant plasmid: pSD107
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: Potassium-transporting ATPase potassium-binding subunit

MacromoleculeName: Potassium-transporting ATPase potassium-binding subunit
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 59.247684 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV ...String:
MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV DLLRITLWVL VPVALLIALF FIQQGALQNF LPYQAVNTVE GAQQLLPMGP VASQEAIKML GTNGGGFFNA NS SHPFENP TALTNFVQML AIFLIPTALC FAFGEVMGDR RQGRMLLWAM SVIFVICVGV VMWAEVQGNP HLLALGTDSS INM EGKESR FGVLVSSLFA VVTTAASCGA VIAMHDSFTA LGGMVPMWLM QIGEVVFGGV GSGLYGMMLF VLLAVFIAGL MIGR TPEYL GKKIDVREMK LTALAILVTP TLVLMGAALA MMTDAGRSAM LNPGPHGFSE VLYAVSSAAN NNGSAFAGLS ANSPF WNCL LAFCMFVGRF GVIIPVMAIA GSLVSKKSQA ASSGTLPTHG PLFVGLLIGT VLLVGALTFI PALALGPVAE YLS

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Macromolecule #2: Potassium-transporting ATPase ATP-binding subunit

MacromoleculeName: Potassium-transporting ATPase ATP-binding subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type K+ transporter
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 72.251867 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE ...String:
MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE SGGDFASVTG GTRILSDWLV IECSVNPGET FLDRMIAMVE GAQRRKTPNE IALTILLIAL TIVFLLATAT LW PFSAWGG NAVSVTVLVA LLVCLIPTTI GGLLSAIGVA GMSRMLGANV IATSGRAVEA AGDVDVLLLD KTGTITLGNR QAS EFIPAQ GVDEKTLADA AQLASLADET PEGRSIVILA KQRFNLRERD VQSLHATFVP FTAQSRMSGI NIDNRMIRKG SVDA IRRHV EANGGHFPTD VDQKVDQVAR QGATPLVVVE GSRVLGVIAL KDIVKGGIKE RFAQLRKMGI KTVMITGDNR LTAAA IAAE AGVDDFLAEA TPEAKLALIR QYQAEGRLVA MTGDGTNDAP ALAQADVAVA MNSGTQAAKE AGNMVDLDSN PTKLIE VVH IGKQMLMTRG SLTTFSIAND VAKYFAIIPA AFAATYPQLN ALNIMCLHSP DSAILSAVIF NALIIVFLIP LALKGVS YK PLTASAMLRR NLWIYGLGGL LVPFIGIKVI DLLLTVCGLV

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Macromolecule #3: Potassium-transporting ATPase KdpC subunit

MacromoleculeName: Potassium-transporting ATPase KdpC subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 22.299225 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV ...String:
MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV KYIGQPVVNI VELNLALDKL DEGTGLVPRG SSHHHHHHHH

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Macromolecule #4: Potassium-transporting ATPase KdpF subunit

MacromoleculeName: Potassium-transporting ATPase KdpF subunit / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 3.071714 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSAGVITGVL LVFLLLGYLV YALINAEAF

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Macromolecule #5: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #6: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)...

MacromoleculeName: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate
type: ligand / ID: 6 / Number of copies: 1 / Formula: 9Y0
Molecular weightTheoretical: 717.996 Da
Chemical component information

ChemComp-9Y0:
(2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 13 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
100.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
1.0 mMC9H15O6Ptris(2-carboxyethyl)phosphine
1.5 mg/mLC22H42O11decyl maltopyranoside
2.5 mMBeF3beryllium fluoride
100.0 mMNaClSodium chloridesodium chloride

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.
GridModel: 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
VitrificationCryogen 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..
DetailsDM-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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1525715 / Details: template based picking
CTF correctionSoftware - Name: cryoSPARC (ver. 2.9.0)
Startup modelType of model: NONE / Details: ab initio
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.9.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 200000 / Software - Name: cryoSPARC (ver. 2.9.0) / Details: hetero refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.0) / Details: non-uniform refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9.0) / Details: non-uniform refinement / Number images used: 441322
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5mrw

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-7bh2:
Cryo-EM Structure of KdpFABC in E2Pi state with BeF3 and K+

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