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- EMDB-1367: Three-dimensional structure of a voltage-gated potassium channel ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1367
TitleThree-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution.
Map dataThis is an image of surface rendered view of Shaker potassium voltage-gated channel
Sample
  • Sample: Shaker B channel
  • Protein or peptide: Kv1.1
Function / homology
Function and homology information


voltage-gated potassium channel activity / voltage-gated potassium channel complex / monoatomic ion transmembrane transport / protein homooligomerization / identical protein binding / plasma membrane
Similarity search - Function
Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Potassium channel domain / Ion channel / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / Voltage-dependent channel domain superfamily / SKP1/BTB/POZ domain superfamily ...Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Potassium channel domain / Ion channel / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / Voltage-dependent channel domain superfamily / SKP1/BTB/POZ domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
pH-gated potassium channel KcsA / Potassium channel
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsSokolova O / Kolmakova-Partensky L / Grigorieff N
CitationJournal: Structure / Year: 2001
Title: Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution.
Authors: O Sokolova / L Kolmakova-Partensky / N Grigorieff /
Abstract: BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial ...BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial homolog, the potassium channel KcsA, is much smaller than Shaker. It does not have a voltage sensor and other important domains like the N-terminal tetramerization (T1) domain. The structure of these additional elements has to be studied in the more complex voltage-gated channels.
RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain ...RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain linked by thin 2 nm long connectors. To interpret the structure, we used the crystal structures of the isolated T1 domain and the KcsA channel. A unique density assignment was made based on the symmetry and dimensions of the crystal structures and domains, identifying the smaller domain as the cytoplasmic mass of Shaker containing T1 and the larger domain as embedded in the membrane.
CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the ...CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the membrane domain but attached to it by thin connectors. The space between the two domains is sufficient to permit cytoplasmic access of ions and the N-terminal inactivation domain to the pore region. A hanging gondola architecture has also been observed in the nicotinic acetylcholine receptor and the KcsA structure, suggesting that it is a common element of ion channels.
History
DepositionMay 24, 2007-
Header (metadata) releaseMay 30, 2007-
Map releaseMay 30, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.531039603
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.531039603
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1367.gif

Downloads & links

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Map

FileDownload / File: emd_1367.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of surface rendered view of Shaker potassium voltage-gated channel
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.5 Å/pix.
x 64 pix.
= 224. Å		3.5 Å/pix.
x 64 pix.
= 224. Å		3.5 Å/pix.
x 64 pix.
= 224. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.5 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.471 / Movie #1: 0.5310396
Minimum - Maximum-0.524587 - 1.17639
Average (Standard dev.)-0.0588283 (±0.213118)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 224 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z224.000224.000224.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-0.5251.176-0.059

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Supplemental data

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Sample components

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Entire : Shaker B channel

EntireName: Shaker B channel
Components
  • Sample: Shaker B channel
  • Protein or peptide: Kv1.1

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Supramolecule #1000: Shaker B channel

SupramoleculeName: Shaker B channel / type: sample / ID: 1000
Details: This is an inactivation-removed Shaker B channel construct, delta6-46/F425G, with a C-terminal 1D4 immunoaffinity tag. The molecular weight given above includes glycosylation.
Oligomeric state: One homotetramer / Number unique components: 1
Molecular weightExperimental: 860 KDa / Theoretical: 860 KDa / Method: SDS PAGE electrophoresis

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Macromolecule #1: Kv1.1

MacromoleculeName: Kv1.1 / type: protein_or_peptide / ID: 1 / Name.synonym: Shaker Kv channel / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Drosophila / Tissue: Muscule / Cell: COS-1 / Organelle: Cell membranes / Location in cell: Cell membranes
Molecular weightExperimental: 860 KDa / Theoretical: 860 KDa
Recombinant expressionOrganism: Mammalian (mammals) / Recombinant plasmid: pmt3

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 80 mM KCl, 2 mM NaEDTA, 40 mM HEPES-KOH, 0.7% CHAPS, 1 mM DTT, 50 mM NaCl, 0.2 mM leupeptin/pepstatin,1 mM PMSF
StainingType: NEGATIVE
Details: Grids floated on drops of sample buffer twice with blotting inbetween, than floated on 1% solution of uranyl acetate twice (blot inbetween) for 10 seconds each
GridDetails: Continuous carbon on 400 mesh copper grid
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.9 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Side-entry room temperature / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 3.5 µm / Number real images: 40 / Average electron dose: 10 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 50
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, FREALIGN / Number images used: 6000
DetailsManual selection of particles

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