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- EMDB-1605: Solution structure of the KdpFABC P-type ATPase from Escherichia ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1605
TitleSolution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis
Map dataVolume of the KdpFABC P-type ATPase
Sample
  • Sample: KdpFABC P-type ATPase
  • Protein or peptide: KdpA-subunit
  • Protein or peptide: KdpB-subunit
  • Protein or peptide: KdpC-subunit
  • Protein or peptide: KdpF-subunit
KeywordsKdpFABC / P-type ATPase / potassium transport / single particle analysis / electron microscopy.
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsHeitkamp T / Bottcher B / Greie J-C
CitationJournal: J Struct Biol / Year: 2009
Title: Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis.
Authors: Thomas Heitkamp / Bettina Böttcher / Jörg-Christian Greie /
Abstract: The K+-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique ...The K+-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique features. It comprises four subunits, and the sites of ATP hydrolysis and substrate transport are located on two different polypeptides. No structural data are so far available for elucidating the correspondingly unique mechanism of coupling ion transport and catalysis in this P-type ATPase. By use of electron microscopy and single particle analysis of negatively stained, solubilized KdpFABC complexes, we solved the structure of the complex at a resolution of 19A, which allowed us to model the arrangement of subunits within the holoenzyme and, thus, to identify the interfaces between subunits. The model showed that the K+-translocating KdpA subunit is in close contact with the transmembrane region of the ATP-hydrolyzing subunit KdpB. The cytosolic C-terminal domain of the KdpC subunit, which is assumed to play a role in cooperative ATP binding together with KdpB, is located in close vicinity to the nucleotide binding domain of KdpB. Overall, the arrangement of subunits agrees with biochemical data and the predictions on subunit interactions.
History
DepositionMar 17, 2009-
Header (metadata) releaseApr 15, 2009-
Map releaseApr 24, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 200
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 200
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image.gif

Downloads & links

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Map

FileDownload / File: emd_1605.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume of the KdpFABC P-type ATPase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 70 pix.
= 196. Å		2.8 Å/pix.
x 70 pix.
= 196. Å		2.8 Å/pix.
x 70 pix.
= 196. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 150.0 / Movie #1: 200
Minimum - Maximum-374.723000000000013 - 1448.240000000000009
Average (Standard dev.)15.9693 (±95.569800000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 196 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z196.000196.000196.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-374.7231448.24115.969

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Supplemental data

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Sample components

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Entire : KdpFABC P-type ATPase

EntireName: KdpFABC P-type ATPase
Components
  • Sample: KdpFABC P-type ATPase
  • Protein or peptide: KdpA-subunit
  • Protein or peptide: KdpB-subunit
  • Protein or peptide: KdpC-subunit
  • Protein or peptide: KdpF-subunit

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Supramolecule #1000: KdpFABC P-type ATPase

SupramoleculeName: KdpFABC P-type ATPase / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightTheoretical: 154 KDa

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Macromolecule #1: KdpA-subunit

MacromoleculeName: KdpA-subunit / type: protein_or_peptide / ID: 1 / Name.synonym: KdpA-subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Molecular weightTheoretical: 59 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGS4

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Macromolecule #2: KdpB-subunit

MacromoleculeName: KdpB-subunit / type: protein_or_peptide / ID: 2 / Name.synonym: KdpB-subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Molecular weightTheoretical: 72 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGS4

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Macromolecule #3: KdpC-subunit

MacromoleculeName: KdpC-subunit / type: protein_or_peptide / ID: 3 / Name.synonym: KdpC-subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Molecular weightTheoretical: 21 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGS4

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Macromolecule #4: KdpF-subunit

MacromoleculeName: KdpF-subunit / type: protein_or_peptide / ID: 4 / Name.synonym: KdpF-subunit / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: membrane
Molecular weightTheoretical: 3 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGS4

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.005 mg/mL
BufferpH: 6
Details: 50 mM MES pH 6.0, 50 mM NaCl, 5 mM MgCl2, 5 mM CaCl2
StainingType: NEGATIVE
Details: applied to freshly glow-discharged carbon-coated copper grids (400 mesh). Staining with 2 % (w/v) uranylacetic acid
GridDetails: 400 mesh copper grid, carbon coated
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 48000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 0.864 µm / Nominal defocus min: 0.432 µm / Nominal magnification: 52000
Sample stageSpecimen holder: room temperature holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 95 K / Max: 295 K / Average: 295 K
Alignment procedureLegacy - Astigmatism: manually at 200000 on carbon film
DateJul 20, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 90 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC Spider / Number images used: 10040
Detailsstarted with angular reconstitution in IMAGIC (25 class averages) followed by projection matching in Spider (global search in 10 degree steps followed by local search in 2 degree steps)

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