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- EMDB-1094: Electron microscopic analysis of KvAP voltage-dependent K+ channe... -

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Basic information

Entry
Database: EMDB / ID: EMD-1094
TitleElectron microscopic analysis of KvAP voltage-dependent K+ channels in an open conformation.
Map dataA map of the KvAP/33H1Fab complex at 10.5 angstrom resolution. A threshold at 1.0 gives 340 kDa.
Sample
  • Sample: KvAP complexed with 33H1 Fab fragments
  • Protein or peptide: KvAP potassium channel
  • Protein or peptide: 33H1 Fab fragments
Biological speciesAeropyrum pernix (archaea) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 10.5 Å
AuthorsJiang QX / Wang DN / MacKinnon R
CitationJournal: Nature / Year: 2004
Title: Electron microscopic analysis of KvAP voltage-dependent K+ channels in an open conformation.
Authors: Qiu-Xing Jiang / Da-Neng Wang / Roderick MacKinnon /
Abstract: Voltage-dependent ion channels serve as field-effect transistors by opening a gate in response to membrane voltage changes. The gate's response to voltage is mediated by voltage sensors, which are ...Voltage-dependent ion channels serve as field-effect transistors by opening a gate in response to membrane voltage changes. The gate's response to voltage is mediated by voltage sensors, which are arginine-containing structures that must move with respect to the membrane electric field. We have analysed by electron microscopy a voltage-dependent K(+) channel from Aeropyrum pernix (KvAP). Fab fragments were attached to 'voltage sensor paddles' and identified in the electron microscopy map at 10.5 A resolution. The extracellular surface location of the Fab fragments in the map is consistent with the membrane-depolarized, open conformation of the channel in electrophysiological experiments. Comparison of the map with a crystal structure demonstrates that the voltage sensor paddles are 'up' (that is, near the channel's extracellular surface) and situated at the protein-lipid interface. This finding supports the hypothesis that in response to changes in voltage the sensors move at the protein-lipid interface rather than in a gating pore surrounded by protein.
History
DepositionMay 26, 2004-
Header (metadata) releaseAug 24, 2004-
Map releaseSep 24, 2004-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.45
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1094.gif

Downloads & links

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Map

FileDownload / File: emd_1094.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA map of the KvAP/33H1Fab complex at 10.5 angstrom resolution. A threshold at 1.0 gives 340 kDa.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 60 pix.
= 168. Å		2.8 Å/pix.
x 60 pix.
= 168. Å		2.8 Å/pix.
x 60 pix.
= 168. 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 Level1: 2.12 / Movie #1: 1.45
Minimum - Maximum-10.7372 - 8.15827
Average (Standard dev.)0.0763837 (±1.00394)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-30-30
Dimensions606060
Spacing606060
CellA=B=C: 168 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z168.000168.000168.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-96-56-288
NX/NY/NZ192112576
MAP C/R/S123
start NC/NR/NS-30-30-30
NC/NR/NS606060
D min/max/mean-10.7378.1580.076

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Supplemental data

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Sample components

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Entire : KvAP complexed with 33H1 Fab fragments

EntireName: KvAP complexed with 33H1 Fab fragments
Components
  • Sample: KvAP complexed with 33H1 Fab fragments
  • Protein or peptide: KvAP potassium channel
  • Protein or peptide: 33H1 Fab fragments

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Supramolecule #1000: KvAP complexed with 33H1 Fab fragments

SupramoleculeName: KvAP complexed with 33H1 Fab fragments / type: sample / ID: 1000 / Details: none / Oligomeric state: one / Number unique components: 2
Molecular weightExperimental: 340 KDa / Theoretical: 300 KDa
Method: the sequence and the estimate of detergent micelle size

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Macromolecule #1: KvAP potassium channel

MacromoleculeName: KvAP potassium channel / type: protein_or_peptide / ID: 1 / Name.synonym: KvAP / Details: one channel / Number of copies: 1 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Aeropyrum pernix (archaea) / synonym: A. pernix / Location in cell: plasma membrane
Molecular weightExperimental: 120 KDa / Theoretical: 120 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pQE60

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Macromolecule #2: 33H1 Fab fragments

MacromoleculeName: 33H1 Fab fragments / type: protein_or_peptide / ID: 2 / Name.synonym: 33H1Fab / Details: four Fab fragments / Number of copies: 4 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse
Molecular weightExperimental: 180 KDa / Theoretical: 180 KDa

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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

Concentration5.0 mg/mL
BufferpH: 7.4
Details: 40 mM KCl, 60 mM NaCl, 20 mM Tris-HCl pH 7.4, 5.0 mM DM
StainingType: NEGATIVE / Details: mix with equal volume of 65% ammonium molybdate
GridDetails: 400 mesh grids with holey carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made / Method: 8

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Electron microscopy

MicroscopeFEI/PHILIPS CM200T
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: corrected at 200000
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 37 / Average electron dose: 20 e/Å2 / Od range: 1.2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.1 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Oxford / Specimen holder model: OTHER

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Image processing

Detailsthe holes in the carbon film were covered with a thin-layer of carbon film.
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC SPIDER / Number images used: 21379
Final two d classificationNumber classes: 496

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