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- EMDB-5114: BK channel with membrane density subtracted -

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Basic information

Entry
Database: EMDB / ID: EMD-5114
TitleBK channel with membrane density subtracted
Map dataSurface rendering of the inside-out BK channel map with membrane density subtracted. The mesh shows a membrane-patch difference map.
Sample
  • Sample: hSlo protein was reconstituted into POPC liposomes.
  • Protein or peptide: hSlo
KeywordsPotassium channel
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsWang L / Sigworth FJ
CitationJournal: 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
DepositionApr 1, 2009-
Header (metadata) releaseJul 31, 2009-
Map releaseJul 31, 2009-
UpdateSep 10, 2009-
Current statusSep 10, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.23
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5114_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5114.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSurface rendering of the inside-out BK channel map with membrane density subtracted. The mesh shows a membrane-patch difference map.
Voxel sizeX=Y=Z: 2.5334 Å
Density
Contour LevelBy AUTHOR: 0.229 / Movie #1: 0.23
Minimum - Maximum-0.295631 - 1.02224
Average (Standard dev.)0.0202579 (±0.120427)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 243.206 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.53339583333332.53339583333332.5333958333333
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z243.206243.206243.206
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.2961.0220.020

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Supplemental data

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Sample components

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Entire : hSlo protein was reconstituted into POPC liposomes.

EntireName: hSlo protein was reconstituted into POPC liposomes.
Components
  • Sample: hSlo protein was reconstituted into POPC liposomes.
  • Protein or peptide: hSlo

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Supramolecule #1000: hSlo protein was reconstituted into POPC liposomes.

SupramoleculeName: hSlo protein was reconstituted into POPC liposomes. / type: sample / ID: 1000 / Oligomeric state: tetramer of human Slo1 subunits / Number unique components: 1
Molecular weightExperimental: 500 KDa / Theoretical: 500 KDa

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Macromolecule #1: hSlo

MacromoleculeName: hSlo / type: protein_or_peptide / ID: 1 / Name.synonym: BK channel alpha subunit / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HEK cell line / Location in cell: Plasma membrane
Molecular weightTheoretical: 125 KDa
Recombinant expressionOrganism: HEK 293 cell / Recombinant plasmid: pcDNA3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: Solution outside proteoliposomes, 13.5mM KCl, 0.5mM NaCl, 0.1mM EDTA, 2mM Hepes
GridDetails: home-made holey carbon film
VitrificationCryogen name: ETHANE / Instrument: OTHER / Method: Manual blot for 2-4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 30.0 eV
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
DateJul 15, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final angle assignmentDetails: phi 90degrees, theta 180 degrees, psi 360 degrees
Final reconstructionAlgorithm: 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 phi according to the spherical geometry of lipid vesicles.
Number images used: 3400

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