+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5121 | |||||||||
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Title | BK channel with membrane density restored | |||||||||
Map data | 3D reconstruction of BK channel with membrane restored | |||||||||
Sample |
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Keywords | Potassium channel | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 17.0 Å | |||||||||
Authors | Wang L / Sigworth FJ | |||||||||
Citation | Journal: Nature / Year: 2009 Title: Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Authors: Liguo Wang / Fred J Sigworth / Abstract: A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases ...A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5121.map.gz | 3.1 MB | EMDB map data format | |
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Header (meta data) | emd-5121-v30.xml emd-5121.xml | 9.4 KB 9.4 KB | Display Display | EMDB header |
Images | emd_5121_1.jpg | 48.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5121 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5121 | HTTPS FTP |
-Validation report
Summary document | emd_5121_validation.pdf.gz | 78 KB | Display | EMDB validaton report |
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Full document | emd_5121_full_validation.pdf.gz | 77.1 KB | Display | |
Data in XML | emd_5121_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5121 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5121 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5121.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of BK channel with membrane restored | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.5334 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : hSlo protein was reconstituted into POPC liposomes.
Entire | Name: hSlo protein was reconstituted into POPC liposomes. |
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Components |
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-Supramolecule #1000: hSlo protein was reconstituted into POPC liposomes.
Supramolecule | Name: hSlo protein was reconstituted into POPC liposomes. / type: sample / ID: 1000 / Oligomeric state: tetramer of human Slo1 subunits / Number unique components: 1 |
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Molecular weight | Experimental: 500 KDa / Theoretical: 500 KDa |
-Macromolecule #1: hSlo
Macromolecule | Name: hSlo / type: protein_or_peptide / ID: 1 / Name.synonym: BK channel alpha subunit / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human / Cell: HEK293 / Location in cell: Plasma membrane |
Molecular weight | Theoretical: 125 KDa |
Recombinant expression | Organism: HEK293 / Recombinant plasmid: pcDNA3 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 Details: Solution outside proteoliposomes, 13.5mM KCl, 0.5mM NaCl, 0.1mM EDTA, 2mM Hepes |
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Grid | Details: Homemade holey carbon film |
Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Homemade plunger / Method: Blot for 2-4 seconds before plunging |
-Electron microscopy
Microscope | FEI TECNAI F30 |
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Temperature | Average: 93 K |
Specialist optics | Energy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 30.0 eV |
Date | Jul 15, 2008 |
Image recording | Category: CCD / Film or detector model: GENERIC CCD / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F30 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each micrograph |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Homemade Matlab code Details: Alignment and reconstruction were modeled on Frealign but with constraints on theta and psi according to the spherical geometry of lipid vesicles. Number images used: 3400 |
Final angle assignment | Details: phi 90 degreees, theta 180 degrees, psi 360 degrees |