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- EMDB-12478: Cryo-EM structure of the KdpFABC complex in an E1-ATP conformatio... -

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Entry
Database: EMDB / ID: EMD-12478
TitleCryo-EM structure of the KdpFABC complex in an E1-ATP conformation loaded with K+
Map dataKdpFABC loaded with K in a E1-ATP state at 3.1 A resolution, sharpened at -92 A2. Data obtained in presence of 5 mM AMPPCP and 50 mM KCl. Contains KdpB D307N mutation.
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
  • Ligand: POTASSIUM ION
  • Ligand: CARDIOLIPIN
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
KeywordsP-type ATPase / superfamily of K+ transporters (SKT) / potassium uptake system / ATP analogue / MEMBRANE PROTEIN
Function / homology
Function and homology information


plasma membrane => GO:0005886 / 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 / hydrolase activity ...plasma membrane => GO:0005886 / 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 / hydrolase activity / 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 ATP-binding subunit / Potassium-transporting ATPase KdpC subunit / Potassium-transporting ATPase potassium-binding subunit / Potassium-transporting ATPase KdpF subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSilberberg JM / Corey RA
Funding support Netherlands, Germany, United Kingdom, 5 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
German Research Foundation (DFG)6322/3 Germany
German Research Foundation (DFG)CRC807 Germany
Wellcome Trust208361/Z/17/Z United Kingdom
Citation
Journal: Nat Commun / Year: 2021
Title: Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC.
Authors: Jakob M Silberberg / Robin A Corey / Lisa Hielkema / Charlott Stock / Phillip J Stansfeld / Cristina Paulino / Inga Hänelt /
Abstract: KdpFABC, a high-affinity K pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD ...KdpFABC, a high-affinity K pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.
#1: Journal: Biorxiv / Year: 2021
Title: Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC
Authors: Silberberg JM / Corey RA / Hielkema L / Stock C / Stansfeld PJ / Paulino C / Hanelt I
History
DepositionFeb 25, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nnl
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12478.png

Downloads & links

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Map

FileDownload / File: emd_12478.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKdpFABC loaded with K in a E1-ATP state at 3.1 A resolution, sharpened at -92 A2. Data obtained in presence of 5 mM AMPPCP and 50 mM KCl. Contains KdpB D307N mutation.
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: 0.0325 / Movie #1: 0.035
Minimum - Maximum-0.15485522 - 0.2671655
Average (Standard dev.)0.00047610703 (±0.0076365476)
SymmetrySpace group: 1
Details

EMDB XML:

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

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z242.880242.880242.880
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1550.2670.000

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Supplemental data

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Mask #1

Fileemd_12478_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_12478_half_map_1.map
Annotationhalf map 1 used during refinement and for FSC gold-standard resolution calculation KdpFABC E1-ATP
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_12478_half_map_2.map
Annotationhalf map 2 used during refinement and for FSC gold-standard resolution calculation KdpFABC E1-ATP
Projections & Slices
AxesZYX

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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
  • Ligand: POTASSIUM ION
  • Ligand: CARDIOLIPIN
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

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Supramolecule #1: KdpFABC

SupramoleculeName: KdpFABC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
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

UniProtKB: Potassium-transporting ATPase potassium-binding subunit

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Macromolecule #2: Potassium-transporting ATPase KdpC subunit

MacromoleculeName: Potassium-transporting ATPase KdpC subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
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

UniProtKB: Potassium-transporting ATPase KdpC subunit

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Macromolecule #3: Potassium-transporting ATPase KdpF subunit

MacromoleculeName: Potassium-transporting ATPase KdpF subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.853463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSAGVITGVL LVFLLLGYLV YALINAE

UniProtKB: Potassium-transporting ATPase KdpF subunit

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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 / Details: S162-P / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type K+ transporter
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 72.346859 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 E(SEP)APVIRE ...String:
MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G E(SEP)APVIRE SGGDFASVTG GTRILSDWLV IECSVNPGET FLDRMIAMVE GAQRRKTPNE IALTILLIAL TIVFLLAT A TLWPFSAWGG NAVSVTVLVA LLVCLIPTTI GGLLSAIGVA GMSRMLGANV IATSGRAVEA AGDVDVLLLN 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

UniProtKB: Potassium-transporting ATPase ATP-binding subunit

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Macromolecule #5: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 10 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #6: CARDIOLIPIN

MacromoleculeName: CARDIOLIPIN / type: ligand / ID: 6 / Number of copies: 2 / Formula: CDL
Molecular weightTheoretical: 1.464043 KDa
Chemical component information

ChemComp-CDL:
CARDIOLIPIN / phospholipid*YM

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Macromolecule #7: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: ACP
Molecular weightTheoretical: 505.208 Da
Chemical component information

ChemComp-ACP:
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / AMP-PCP, energy-carrying molecule analogue*YM

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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

Concentration3.1 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: 6 / Number real images: 5831 / 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: 331673
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6hra

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 160776
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

Initial modelPDB ID:

6hra


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-7nnl:
Cryo-EM structure of the KdpFABC complex in an E1-ATP conformation loaded with K+

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