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- EMDB-1074: Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels b... -

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Basic information

Entry
Database: EMDB / ID: EMD-1074
TitleThree-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution.
Map dataThis is the 3D map of the Kv4.2-KChIP complex
Sample
  • Sample: Human Kv4.2-KChIP2 Ion Channel Complex
  • Protein or peptide: Kv4.2
  • Ligand: KChIP2
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsKim LA / Furst J / Gutierrez D / Butler MH / Xu S / Goldstein SAN / Grigorieff N
CitationJournal: Neuron / Year: 2004
Title: Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution.
Authors: Leo A Kim / Johannes Furst / David Gutierrez / Margaret H Butler / Shuhua Xu / Steve A N Goldstein / Nikolaus Grigorieff /
Abstract: Regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac I(to) and neuronal I(A) subtypes. Here, ...Regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac I(to) and neuronal I(A) subtypes. Here, negative stain electron microscopy (EM) and single particle averaging reveal KChIP2 to create a novel approximately 35 x 115 x 115 Angstrom, intracellular fenestrated rotunda: four peripheral columns that extend down from the membrane-embedded portion of the channel to enclose the Kv4.2 "hanging gondola" (a platform held beneath the transmembrane conduction pore by four internal columns). To reach the pore from the cytosol, ions traverse one of four external fenestrae to enter the rotundal vestibule and then cross one of four internal windows in the gondola.
History
DepositionMar 23, 2004-
Header (metadata) releaseMar 23, 2004-
Map releaseMar 23, 2004-
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: 1.746453
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.746453
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1074.gif

Downloads & links

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Map

FileDownload / File: emd_1074.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D map of the Kv4.2-KChIP complex
Voxel sizeX=Y=Z: 4.67 Å
Density
Contour Level1: 0.244 / Movie #1: 1.746453
Minimum - Maximum-0.928056 - 2.72242
Average (Standard dev.)0.0213089 (±0.222702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 420.3 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.674.674.67
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z420.300420.300420.300
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-0.9282.7220.021

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Supplemental data

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Sample components

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Entire : Human Kv4.2-KChIP2 Ion Channel Complex

EntireName: Human Kv4.2-KChIP2 Ion Channel Complex
Components
  • Sample: Human Kv4.2-KChIP2 Ion Channel Complex
  • Protein or peptide: Kv4.2
  • Ligand: KChIP2

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Supramolecule #1000: Human Kv4.2-KChIP2 Ion Channel Complex

SupramoleculeName: Human Kv4.2-KChIP2 Ion Channel Complex / type: sample / ID: 1000
Details: The sample was solubilised in 0.7% CHAPS and was monodisperse
Oligomeric state: 4 Kv4.2 subunits bind to 4 KChIP2 subunits
Number unique components: 2
Molecular weightExperimental: 440 KDa / Theoretical: 400 KDa / Method: SDS-PAGE

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Macromolecule #1: Kv4.2

MacromoleculeName: Kv4.2 / type: protein_or_peptide / ID: 1 / Details: 4 monomers form a membrane-embedded channel / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cell membrane
Molecular weightExperimental: 72 MDa / Theoretical: 75 MDa
Recombinant expressionOrganism: COS7 / Recombinant plasmid: pRAT

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Macromolecule #2: KChIP2

MacromoleculeName: KChIP2 / type: ligand / ID: 2
Details: 4 monomers bind on the cytoplasmic side of the channel
Number of copies: 4 / Oligomeric state: Monomer / Recombinant expression: Yes
Molecular weightExperimental: 28 MDa / Theoretical: 35 MDa
Recombinant expressionOrganism: COS7 / Recombinant plasmid: pRAT

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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

Concentration0.1 mg/mL
BufferpH: 7.4
Details: 0.7% CHAPS, 100 mM NaCl, 40 mM KCl, 0.01 mM leupeptin/pepstatin, 1 mM EDTA, 20 mM HEPES-KOH (pH 7.4)
StainingType: NEGATIVE
Details: Grids with adsorbed protein were washed 3 times in detergent-free buffer and then floated on 1% uranyl acetate for 10 seconds
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE

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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: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric, room temperature / Specimen holder model: OTHER
TemperatureAverage: 300 K
Alignment procedureLegacy - Astigmatism: carbon film grain
DateJun 1, 2003
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 4.67 µm / Number real images: 63 / Average electron dose: 10 e/Å2
Details: Images were scanned at 7 microns resolution and 4 x 4 pixels were averaged
Od range: 1.4 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each particle
Final angle assignmentDetails: theta 90 degrees, phi 90 degrees
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, FREALIGN / Details: FREALIGN reconstruction in Fourier space / Number images used: 8164
Detailsparticles were selected manually

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Atomic model buiding 1

Initial model(PDB ID:

1g8i

,

1orq

,

1exb

)
SoftwareName: SITUS
DetailsProtocol: Rigid body. Automated fitting by SITUS, manual fitting using CHIMERA
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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