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- PDB-6nd0: human BK channel reconstituted into liposomes -

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Basic information

Entry
Database: PDB / ID: 6nd0
Titlehuman BK channel reconstituted into liposomes
ComponentsCalcium-activated potassium channel subunit alpha-1
KeywordsMEMBRANE PROTEIN / the large conductance voltage- and calcium-activated potassium (BK) channel / Ca2+ sensor / gating ring / random spherically constrained (RSC) single-particle cryo-EM
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWang, L. / Tonggu, L.
Funding support United States, 1items
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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Calcium-activated potassium channel subunit alpha-1
B: Calcium-activated potassium channel subunit alpha-1
C: Calcium-activated potassium channel subunit alpha-1
D: Calcium-activated potassium channel subunit alpha-1


Theoretical massNumber of molelcules
Total (without water)397,0274
Polymers397,0274
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Calcium-activated potassium channel subunit alpha-1 / BK channel / BKCA alpha / Calcium-activated potassium channel / subfamily M subunit alpha-1 / K(VCA) ...BK channel / BKCA alpha / Calcium-activated potassium channel / subfamily M subunit alpha-1 / K(VCA)alpha / KCa1.1 / Maxi K channel / MaxiK / Slo-alpha / Slo1 / Slowpoke homolog / hSlo


Mass: 99256.867 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNMA1, KCNMA, SLO / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q12791

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human BK channel reconstituted into liposomes / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
112 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
290 mMPotassium chlorideKCl1
31.2 mMEthylenediaminetetraacetic acidEDTA1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Swelled BK proteoliposomes yielded a concentration of 2 mM lipid and 1mg/ml protein.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: The Coma free alignment was performed using Auto-CTF software from FEI.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 852 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 103.5 K / Temperature (min): 90.8 K
Image recordingAverage exposure time: 8.6 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 3089
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.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 43 / Used frames/image: 1-43

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2.1particle selection
2MATLABR2018aparticle selectionHomemade MATLAB programs were used to exclude particles
3Leginon3.3image acquisitionLeginon was used to collected images
5CTFFIND4CTF correction
6GctfCTF correction
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
Image processingDetails: Dose-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 ...Details: Dose-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.
CTF correctionDetails: CTF full correction was performed following RELION 3D reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122456 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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