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- EMDB-14347: Cryo-EM map of the WT KdpFABC complex in the E2-P conformation, s... -

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Basic information

Entry
Database: EMDB / ID: EMD-14347
TitleCryo-EM map of the WT KdpFABC complex in the E2-P conformation, stabilised with the inhibitor orthovanadate
Map dataCryo-EM map of the WT KdpFABC complex, in the E2-P tight conformation, at 7.4 A resolution, sharpened at -195 A2. Data obtained in the presence of 2mM orthovanadate and 1mM KCl.
Sample
  • Complex: KdpFABC
    • Protein or peptide: Potassium-transporting ATPase potassium-binding subunit
    • Protein or peptide: Potassium-transporting ATPase KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit
KeywordsP-type ATPase / superfamily of K+ transporters (SKT) / potassium uptake system / MEMBRANE PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsHielkema L / Stock C / Silberberg JM / Corey RA / Wunnicke D / 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
DepositionFeb 14, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14347.png

Downloads & links

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Map

FileDownload / File: emd_14347.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the WT KdpFABC complex, in the E2-P tight conformation, at 7.4 A resolution, sharpened at -195 A2. Data obtained in the presence of 2mM orthovanadate and 1mM KCl.
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: 0.0215
Minimum - Maximum-0.010023706 - 0.03955991
Average (Standard dev.)0.00053264847 (±0.0037940776)
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_14347_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_14347_half_map_1.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation of the WT KdpFABC complex in the presence of orthovanadate.
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_14347_half_map_2.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation of the WT KdpFABC complex in the presence of orthovanadate.
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 KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit

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Supramolecule #1: KdpFABC

SupramoleculeName: KdpFABC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
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 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGL NMLGLAVLFF MLLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWQSYS G ETTLSYFS QMAGLTVQNF LSAASGIAVI FALIRAFTRQ SMSTLGNAWV ...String:
MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGL NMLGLAVLFF MLLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWQSYS G ETTLSYFS QMAGLTVQNF LSAASGIAVI FALIRAFTRQ SMSTLGNAWV DLLRITLWVL VP VALLIAL FFIQQGALQN FLPYQAVNTV EGAQQLLPMG PVASQEAIKM LGTNGGGFFN ANS SHPFEN PTALTNFVQM LAIFLIPTAL CFAFGEVMGD RRQGRMLLWA MSVIFVICVG VVMW AEVQG NPHLLALGTD SSINMEGKES RFGVLVSSLF AVVTTAASCG AVIAMHDSFT ALGGM VPMW LMQIGEVVFG GVGSGLYGMM LFVLLAVFIA GLMIGRTPEY LGKKIDVREM KLTALA ILV TPTLVLMGAA LAMMTDAGRS AMLNPGPHGF SEVLYAVSSA ANNNGSAFAG LSANSPF WN CLLAFCMFVG RFGVIIPVMA IAGSLVSKKS QAASSGTLPT HGPLFVGLLI GTVLLVGA L TFIPALALGP VAEYLS

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Macromolecule #2: Potassium-transporting ATPase KdpC subunit

MacromoleculeName: Potassium-transporting ATPase KdpC subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRP SATAEMPYNP QASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE L VTASASGL DNNITPQAAA WQIPRVAKAR NLSVEQLTQL IAKYSQQPLV KYIGQPVVNI VE LNLALDK LDE

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Macromolecule #3: Potassium-transporting ATPase KdpF subunit

MacromoleculeName: Potassium-transporting ATPase KdpF subunit / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSAGVITGVL LVFLLLGYLV YALINAE

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Macromolecule #4: Potassium-transporting ATPase ATP-binding subunit

MacromoleculeName: Potassium-transporting ATPase ATP-binding subunit / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO / EC number: P-type K+ transporter
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAIS GWLWITVLFA NFAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD K VPADQLRK GDIVLVEAGD IIPCDGEVIE GGASVDESAI TGE(SEP)APVIRE ...String:
MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAIS GWLWITVLFA NFAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD K VPADQLRK GDIVLVEAGD IIPCDGEVIE GGASVDESAI TGE(SEP)APVIRE SGGDFASVTG GT RILSDWL VIECSVNPGE TFLDRMIAMV EGAQRRKTPN EIALTILLIA LTIVFLLATA TLW PFSAWG GNAVSVTVLV ALLVCLIPTT IGGLLSAIGV AGMSRMLGAN VIATSGRAVE AAGD VDVLL LDKTGTITLG NRQASEFIPA QGVDEKTLAD AAQLASLADE TPEGRSIVIL AKQRF NLRE RDVQSLHATF VPFTAQSRMS GINIDNRMIR KGSVDAIRRH VEANGGHFPT DVDQKV DQV ARQGATPLVV VEGSRVLGVI ALKDIVKGGI KERFAQLRKM GIKTVMITGD NRLTAAA IA AEAGVDDFLA EATPEAKLAL IRQYQAEGRL VAMTGDGTND APALAQADVA VAMNSGTQ A AKEAGNMVDL DSNPTKLIEV VHIGKQMLMT RGSLTTFSIA NDVAKYFAII PAAFAATYP QLNALNIMCL HSPDSAILSA VIFNALIIVF LIPLALKGVS YKPLTASAML RRNLWIYGLG GLLVPFIGI KVIDLLLTVC GLV

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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

Concentration4.0 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 / Pretreatment - Time: 20 sec. / 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: 2 / Number real images: 2487 / 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: 164891
Startup modelType of model: PDB ENTRY
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 13508
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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