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- EMDB-14918: Cryo-EM map of the unphosphorylated KdpFABC complex in the E2-P c... -

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Basic information

Entry
Database: EMDB / ID: EMD-14918
TitleCryo-EM map of the unphosphorylated KdpFABC complex in the E2-P conformation, under turnover conditions
Map dataCryo-EM map of the unphosphorylated KdpFABC complex, in the E2-P conformation, at 4.0 A resolution, sharpened at -146 A2. Data obtained in the presence under turnover conditions
Sample
  • Complex: KdpFABC
    • 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 ION
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
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: 4.0 Å
AuthorsHielkema L / Stock C / Silberberg JM / Corey RA / Wunnicke D / Dubach VRA / Stansfeld PJ / Haenelt I / Paulino C
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 United Kingdom
Netherlands Organisation for Scientific Research (NWO)740.018.016 United Kingdom
German Research Foundation (DFG)6322/3 United Kingdom
German Research Foundation (DFG)CRC807 United Kingdom
Wellcome Trust208361/Z/17/Z United Kingdom
CitationJournal: 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
DepositionMay 4, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14918.png

Downloads & links

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Map

FileDownload / File: emd_14918.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the unphosphorylated KdpFABC complex, in the E2-P conformation, at 4.0 A resolution, sharpened at -146 A2. Data obtained in the presence under turnover conditions
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 242.88 Å		1.01 Å/pix.
x 240 pix.
= 242.88 Å		1.01 Å/pix.
x 240 pix.
= 242.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.08795208 - 0.13995431
Average (Standard dev.)7.759597e-05 (±0.0061528506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 242.87999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14918_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 used during refinement and for...

Fileemd_14918_half_map_1.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation of the unphosphorylated KdpFABC complex in the E2-P conformation under turnover conditions
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 used during refinement and for...

Fileemd_14918_half_map_2.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation of the unphosphorylated KdpFABC complex in the E2-P conformation under turnover conditions
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

EntireName: KdpFABC
Components
  • Complex: KdpFABC
    • 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 ION

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Supramolecule #1: KdpFABC

SupramoleculeName: KdpFABC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: LB2003
Molecular weightTheoretical: 157 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: 59.218613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWQSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV ...String:
MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWQSYS 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) / Strain: K12
Molecular weightTheoretical: 72.331844 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 AGDVDVLLL(PHD) KTGTITLGN RQASEFIPAQ GVDEKTLADA AQLASLADET PEGRSIVILA KQRFNLRERD VQSLHATFVP FTAQSRMSGI NIDNRMIRKG SVDAIRRHV EANGGHFPTD VDQKVDQVAR QGATPLVVVE GSRVLGVIAL KDIVKGGIKE RFAQLRKMGI KTVMITGDNR L TAAAIAAE AGVDDFLAEA TPEAKLALIR QYQAEGRLVA MTGDGTNDAP ALAQADVAVA MNSGTQAAKE AGNMVDLDSN PT KLIEVVH IGKQMLMTRG SLTTFSIAND VAKYFAIIPA AFAATYPQLN ALNIMCLHSP DSAILSAVIF NALIIVFLIP LAL KGVSYK 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) / Strain: K12
Molecular weightTheoretical: 20.281035 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 KYIGQPVVNI VELNLALDKL DE

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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: 2.853463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSAGVITGVL LVFLLLGYLV YALINAE

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Macromolecule #5: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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

Concentration3.4 mg/mL
BufferpH: 8
Details: 10 mM Tris-HCl pH 8, 10 mM MgCl2, 10 mM NaCl and 0.0125% DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: at 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 3 / Number real images: 11428 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 49407
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 287232
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6hrb

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 47004
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6hrb

RefinementSpace: REAL
Output model

PDB-7zrl:
Cryo-EM map of the unphosphorylated KdpFABC complex in the E2-P conformation, under turnover conditions

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