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- PDB-8gh9: Cryo-EM structure of hSlo1 in total membrane vesicles -

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Basic information

Entry
Database: PDB / ID: 8gh9
TitleCryo-EM structure of hSlo1 in total membrane vesicles
ComponentsCalcium-activated potassium channel subunit alpha-1
KeywordsTRANSPORT PROTEIN / Slo1 / BK channel / Ca2+- and voltage-activated K+ channel / ion channel / toal cell membrane vesicles
Function / homology
Function and homology information


Acetylcholine inhibits contraction of outer hair cells / large conductance calcium-activated potassium channel activity / micturition / Ca2+ activated K+ channels / response to carbon monoxide / calcium-activated potassium channel activity / negative regulation of cell volume / intracellular potassium ion homeostasis / smooth muscle contraction involved in micturition / Sensory processing of sound by inner hair cells of the cochlea ...Acetylcholine inhibits contraction of outer hair cells / large conductance calcium-activated potassium channel activity / micturition / Ca2+ activated K+ channels / response to carbon monoxide / calcium-activated potassium channel activity / negative regulation of cell volume / intracellular potassium ion homeostasis / smooth muscle contraction involved in micturition / Sensory processing of sound by inner hair cells of the cochlea / response to osmotic stress / cGMP effects / voltage-gated potassium channel activity / voltage-gated potassium channel complex / potassium ion transmembrane transport / regulation of membrane potential / caveola / potassium ion transport / response to calcium ion / vasodilation / actin binding / postsynaptic membrane / response to hypoxia / positive regulation of apoptotic process / apical plasma membrane / membrane / identical protein binding / 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: 3.8 Å
AuthorsTao, X. / Zhao, C. / MacKinnon, R.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Jane Coffin Childs (JCC) Fund United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Membrane protein isolation and structure determination in cell-derived membrane vesicles.
Authors: Xiao Tao / Chen Zhao / Roderick MacKinnon /
Abstract: Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of ...Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.
History
DepositionMar 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)483,6334
Polymers483,6334
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron 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 / Slo homolog / hSlo


Mass: 120908.125 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNMA1, KCNMA, SLO / 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: ALFA-hSlo1 tetrameric channel / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 0.001 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 21348

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
1Topazparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11RELIONclassification
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85135 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00427688
ELECTRON MICROSCOPYf_angle_d0.73937567
ELECTRON MICROSCOPYf_dihedral_angle_d4.6513618
ELECTRON MICROSCOPYf_chiral_restr0.0454302
ELECTRON MICROSCOPYf_plane_restr0.0044717

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