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- EMDB-1094: Electron microscopic analysis of KvAP voltage-dependent K+ channe... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-1094 | |||||||||
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Title | Electron microscopic analysis of KvAP voltage-dependent K+ channels in an open conformation. | |||||||||
![]() | A map of the KvAP/33H1Fab complex at 10.5 angstrom resolution. A threshold at 1.0 gives 340 kDa. | |||||||||
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Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 10.5 Å | |||||||||
![]() | Jiang QX / Wang DN / MacKinnon R | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 599.2 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.6 KB 10.6 KB | Display Display | ![]() |
Images | ![]() | 36.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 238.6 KB | Display | ![]() |
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Full document | ![]() | 237.6 KB | Display | |
Data in XML | ![]() | 4.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | A map of the KvAP/33H1Fab complex at 10.5 angstrom resolution. A threshold at 1.0 gives 340 kDa. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : KvAP complexed with 33H1 Fab fragments
Entire | Name: KvAP complexed with 33H1 Fab fragments |
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Components |
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-Supramolecule #1000: KvAP complexed with 33H1 Fab fragments
Supramolecule | Name: KvAP complexed with 33H1 Fab fragments / type: sample / ID: 1000 / Details: none / Oligomeric state: one / Number unique components: 2 |
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Molecular weight | Experimental: 340 KDa / Theoretical: 300 KDa Method: the sequence and the estimate of detergent micelle size |
-Macromolecule #1: KvAP potassium channel
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Experimental: 120 KDa / Theoretical: 120 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: 33H1 Fab fragments
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 180 KDa / Theoretical: 180 KDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 5.0 mg/mL |
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Buffer | pH: 7.4 Details: 40 mM KCl, 60 mM NaCl, 20 mM Tris-HCl pH 7.4, 5.0 mM DM |
Staining | Type: NEGATIVE / Details: mix with equal volume of 65% ammonium molybdate |
Grid | Details: 400 mesh grids with holey carbon film |
Vitrification | Cryogen 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
Microscope | FEI/PHILIPS CM200T |
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Temperature | Average: 100 K |
Alignment procedure | Legacy - Astigmatism: corrected at 200000 |
Image recording | Category: 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 min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination 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 stage | Specimen holder: Oxford / Specimen holder model: OTHER |
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Image processing
Details | the holes in the carbon film were covered with a thin-layer of carbon film. |
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CTF correction | Details: each particle |
Final reconstruction | Applied 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 classification | Number classes: 496 |