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- PDB-6v3g: Cryo-EM structure of Ca2+-free hsSlo1 channel -

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Basic information

Entry
Database: PDB / ID: 6v3g
TitleCryo-EM structure of Ca2+-free hsSlo1 channel
ComponentsCalcium-activated potassium channel subunit alpha-1
KeywordsTRANSPORT PROTEIN / High conductance Ca2+-activated K+ channel / Slo1 channel / BK channel / MaxiK channel
Function / homology
Function and homology information


translation release factor activity, codon specific / cytoplasm
Similarity search - Function
Peptide chain release factor aRF1 / Calcium-activated BK potassium channel alpha subunit / Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 1 ...Peptide chain release factor aRF1 / Calcium-activated BK potassium channel alpha subunit / Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 1 / eRF1 domain 3 / eRF1_1 / 50S ribosomal protein L30e-like / Ion transport protein
Similarity search - Domain/homology
Peptide chain release factor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsTao, X. / MacKinnon, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
CitationJournal: Elife / Year: 2019
Title: Molecular structures of the human Slo1 K channel in complex with β4.
Authors: Xiao Tao / Roderick MacKinnon /
Abstract: Slo1 is a Ca- and voltage-activated K channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by ...Slo1 is a Ca- and voltage-activated K channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, β4. Four β4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, β4 forms a tetrameric crown over the pore. Structures with high and low Ca concentrations show that identical gating conformations occur in the absence and presence of β4, implying that β4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of β4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.
History
DepositionNov 25, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • EMDB-21036
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Calcium-activated potassium channel subunit alpha-1
A: 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)479,9524
Polymers479,9524
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14800 Å2
ΔGint-92 kcal/mol
Surface area155970 Å2

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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: 119988.062 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): HEK293S GnTI- / 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: Ca2+-free hsSlo1 channel / Type: COMPLEX
Details: human high conductance Ca2+-activated K+ channel Slo1 (BK) in the absence of Calcium
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293S GnTI-
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1450 mMpotassium chlorideKCl1
220 mMTrisC4H11NO31
30.5 mMDigitoninC56H92O291
45 mMEGTAC14H24N2O101
515 mMmagnesium chlorideMgCl21
SpecimenConc.: 8.5 mg/ml / Embedding 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 K / Details: Blot for 4 seconds before plunging.

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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 FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 89 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1292
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryFitting-ID
1Gautomatchv0.56_sm61_cu8.0particle selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fitting1
8Coot0.8.9.2model fitting1
10cryoSPARC2.9.0initial Euler assignment
11FREALIGN9.11final Euler assignment
12RELION3.0.8classification
13FREALIGN9.113D reconstruction
14PHENIX1.16model refinement1
27PHENIX1.16model refinement2
CTF correctionType: NONE
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53961 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1RIGID BODY FITREAL
2AB INITIO MODELREAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16V35

6v35

16V351PDBexperimental model
22
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00729264
ELECTRON MICROSCOPYf_angle_d0.94239700
ELECTRON MICROSCOPYf_dihedral_angle_d8.16917300
ELECTRON MICROSCOPYf_chiral_restr0.0554536
ELECTRON MICROSCOPYf_plane_restr0.0075000

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