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- EMDB-0439: human BK channel reconstituted into liposomes -

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Basic information

Entry
Database: EMDB / ID: EMD-0439
Titlehuman BK channel reconstituted into liposomes
Map datahuman BK channel reconstituted into liposomes
Sample
  • Complex: human BK channel reconstituted into liposomes
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1
Keywordsthe large conductance voltage- and calcium-activated potassium (BK) channel / Ca2+ sensor / gating ring / random spherically constrained (RSC) single-particle cryo-EM / MEMBRANE PROTEIN
Function / homology
Function and homology information


Acetylcholine inhibits contraction of outer hair cells / micturition / Ca2+ activated K+ channels / response to carbon monoxide / large conductance calcium-activated potassium channel activity / calcium-activated potassium channel activity / negative regulation of cell volume / smooth muscle contraction involved in micturition / intracellular potassium ion homeostasis / Sensory processing of sound by inner hair cells of the cochlea ...Acetylcholine inhibits contraction of outer hair cells / micturition / Ca2+ activated K+ channels / response to carbon monoxide / large conductance calcium-activated potassium channel activity / calcium-activated potassium channel activity / negative regulation of cell volume / smooth muscle contraction involved in micturition / intracellular potassium ion homeostasis / Sensory processing of sound by inner hair cells of the cochlea / response to osmotic stress / voltage-gated potassium channel activity / cGMP effects / potassium ion transmembrane transport / voltage-gated potassium channel complex / caveola / regulation of membrane potential / potassium ion transport / response to calcium ion / vasodilation / actin binding / postsynaptic membrane / response to hypoxia / positive regulation of apoptotic process / apical plasma membrane / identical protein binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
: / Ca2+-activated K+ channel Slowpoke, TrkA_C like domain / : / Calcium-activated potassium channel BK, alpha subunit / Calcium-activated BK potassium channel alpha subunit / Ion transport domain / Ion transport protein / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Calcium-activated potassium channel subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWang L / Tonggu L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM096458 United States
CitationJournal: To Be Published
Title: Broken symmetry in the human BK channel
Authors: Tonggu L / Wang L
History
DepositionDec 13, 2018-
Header (metadata) releaseJun 26, 2019-
Map releaseJun 17, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0153
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0153
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nd0
  • Surface level: 0.0153
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0439.png

Downloads & links

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Map

FileDownload / File: emd_0439.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman BK channel reconstituted into liposomes
Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 0.0153 / Movie #1: 0.0153
Minimum - Maximum-0.052314363 - 0.09064497
Average (Standard dev.)0.0004835051 (±0.0042055035)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 270.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0561.0561.056
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z270.336270.336270.336
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0520.0910.000

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Supplemental data

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Sample components

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Entire : human BK channel reconstituted into liposomes

EntireName: human BK channel reconstituted into liposomes
Components
  • Complex: human BK channel reconstituted into liposomes
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1

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Supramolecule #1: human BK channel reconstituted into liposomes

SupramoleculeName: human BK channel reconstituted into liposomes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Calcium-activated potassium channel subunit alpha-1

MacromoleculeName: Calcium-activated potassium channel subunit alpha-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.256867 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LKTSNSIKLV NLLSIFISTW LTAAGFIHLV ENSGDPWENF QNNQALTYWE CVYLLMVTMS TVGYGDVYAK TTLGRLFMVF FILGGLAMF ASYVPEIIEL IGNRKKYGGS YSAVSGRKHI VVCGHITLES VSNFLKDFLH KDRDDVNVEI VFLHNISPNL E LEALFKRH ...String:
LKTSNSIKLV NLLSIFISTW LTAAGFIHLV ENSGDPWENF QNNQALTYWE CVYLLMVTMS TVGYGDVYAK TTLGRLFMVF FILGGLAMF ASYVPEIIEL IGNRKKYGGS YSAVSGRKHI VVCGHITLES VSNFLKDFLH KDRDDVNVEI VFLHNISPNL E LEALFKRH FTQVEFYQGS VLNPHDLARV KIESADACLI LANKYCADPD AEDASNIMRV ISIKNYHPKI RIITQMLQYH NK AHLLNIP SWNWKEGDDA ICLAELKLGF IAQSCLAQGL STMLANLFSM RSFIKIEEDT WQKYYLEGVS NEMYTEYLSS AFV GLSFPT VCELCFVKLK LLMIAIEYKS ANRESRILIN PGNHLKIQEG TLGFFIASDA KEVKRAFFYC KACHDDITDP KRIK KCGCK RPKMSIYKRM RRACCFDCGR SERDCSCMSG RVRGNVDTLE RAFPLSSVSV NDCSTSFRAF EDEQPSTLSP KKKQR NGGM RNSPNTSPKL MRHDPLLIPG NDQIDNMDSN VKKYDSTGMF HWCAPKEIEK VILTRSEAAM TVLSGHVVVC IFGDVS SAL IGLRNLVMPL RASNFHYHEL KHIVFVGSIE YLKREWETLH NFPKVSILPG TPLSRADLRA VNINLCDMCV ILSANQN NI DDTSLQDKEC ILASLNIKSM QFDDSIGVLQ ANSQGFTPPG MDRSSPDNSP VHGMLRQPSI TTGVNIPIIT ELVNDTNV Q FLDQDDDDDP DTELYLTQPF ACGTAFAVSV LDSLMSATYF NDNILTLIRT LVTGGATPEL EALIAEENAL RGGYSTPQT LANRDRCRVA QLALLDGPFA DLGDGGCYGD LFCKALKTYN MLCFGIYRLR DAHLSTPSQC TKRYVITNPP YEFELVPTDL IFCL

UniProtKB: Calcium-activated potassium channel subunit alpha-1

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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

Concentration1.0 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
12.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
90.0 mMKClPotassium chloride
1.2 mMEDTAEthylenediaminetetraacetic acid
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.133 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: BK proteoliposomes were applied onto a glow-discharged perforated carbon grid (CFlat R2/2, EMS), and incubated for 5-10 min In an FEI Vitrobot Mark IV (FEI). After incubation, the sample was ...Details: BK proteoliposomes were applied onto a glow-discharged perforated carbon grid (CFlat R2/2, EMS), and incubated for 5-10 min In an FEI Vitrobot Mark IV (FEI). After incubation, the sample was blotted from the side and buffer containing 10 mM HEPES, pH 7.3, 75 mM KCl, and 1 mM EDTA was applied onto the TEM grid to further swell proteoliposomes. Then grids were blotted for 7 s with force 0 and fast frozen in liquid ethane cooled by liquid nitrogen..
DetailsSwelled BK proteoliposomes yielded a concentration of 2 mM lipid and 1mg/ml protein.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 90.8 K / Max: 103.5 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsThe Coma free alignment was performed using Auto-CTF software from FEI.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-43 / Number grids imaged: 3 / Number real images: 3089 / Average exposure time: 8.6 sec. / Average electron dose: 60.0 e/Å2
Details: Images were recorded in an automated fashion on a Gatan K2 Summit (Gatan) detector set to super-resolution mode with a super-resolution pixel size of 0.525 A using Leginon. The dose rate on ...Details: Images were recorded in an automated fashion on a Gatan K2 Summit (Gatan) detector set to super-resolution mode with a super-resolution pixel size of 0.525 A using Leginon. The dose rate on the camera was set to be ~8 counts per physical pixel per second. The total exposure time was 8.6 s (0.2 s/frame), leading to a total accumulate dose of 60 electrons per A2 on the specimen.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.852 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsDose-fractionated super-resolution image stacks were motion-corrected with MotionCorr2. Each frame in the image stack was divided into 5x5 patches for anisotropic image motion correction and dose weighting was carried out to calculate the motion-corrected image.
Particle selectionNumber selected: 275295
Details: Approximately 4,800 particles from 350 micrographs were interactively selected and classified using RELION. Ten representative classes were selected as the templates for automated particle ...Details: Approximately 4,800 particles from 350 micrographs were interactively selected and classified using RELION. Ten representative classes were selected as the templates for automated particle picking using RELION. The automated picked particles were screened using homemade MATLAB (MathWorks) programs to exclude particles more than 80 A away from any liposome, which resulted in 275,295 particles from 2,786 micrographs.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5tji

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 122456
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 100 / Avg.num./class: 2750 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6nd0:
human BK channel reconstituted into liposomes

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