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- PDB-8ghf: cryo-EM structure of hSlo1 in plasma membrane vesicles -

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Basic information

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


translation release factor activity, codon specific / cytoplasm
Similarity search - Function
Peptide chain release factor aRF1 / 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 ...Peptide chain release factor aRF1 / 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
Similarity search - Domain/homology
CHOLESTEROL / Chem-POV / Peptide chain release factor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
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Revision 1.0May 10, 2023Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 10, 2023Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0May 10, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.2May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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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
B: Calcium-activated potassium channel subunit alpha-1
C: Calcium-activated potassium channel subunit alpha-1
D: Calcium-activated potassium channel subunit alpha-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)566,437132
Polymers489,7604
Non-polymers76,676128
Water00
1


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

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Components

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Protein , 1 types, 4 molecules ABCD

#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: 122440.102 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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Non-polymers , 5 types, 128 molecules

#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical...
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 88 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#5: Chemical...
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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-eGFP ion channel / Type: COMPLEX / Entity ID: #1 / 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter slit width: 6 eV

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
1Topazparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
8PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32063 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00232536
ELECTRON MICROSCOPYf_angle_d0.57843992
ELECTRON MICROSCOPYf_dihedral_angle_d14.225444
ELECTRON MICROSCOPYf_chiral_restr0.0424856
ELECTRON MICROSCOPYf_plane_restr0.0055180

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