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- PDB-5tj6: Ca2+ bound aplysia Slo1 -

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Basic information

Entry
Database: PDB / ID: 5tj6
TitleCa2+ bound aplysia Slo1
ComponentsHigh conductance calcium-activated potassium channel
KeywordsMEMBRANE PROTEIN / ion channel / K+ channel / Ca2+ bound / high conductance
Function / homology
Function and homology information


potassium ion transport / monoatomic ion channel activity / 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 / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
: / Chem-PGW / BK channel
Similarity search - Component
Biological speciesAplysia californica (California sea hare)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMacKinnon, R. / Tao, X. / Hite, R.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2017
Title: Cryo-EM structure of the open high-conductance Ca-activated K channel.
Authors: Xiao Tao / Richard K Hite / Roderick MacKinnon /
Abstract: The Ca-activated K channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca renders Slo1 central to ...The Ca-activated K channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca renders Slo1 central to processes that couple electrical signalling to Ca-mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca and Mg at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca-binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.
History
DepositionOct 3, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2017Group: Database references
Revision 1.2Jan 18, 2017Group: Database references
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Oct 3, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.5Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[1][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[1][2] / _atom_sites.fract_transf_matrix[1][3] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[2][3] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _atom_sites.fract_transf_vector[1] / _atom_sites.fract_transf_vector[3] / _cell.Z_PDB
Revision 1.7Mar 13, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Assembly

Deposited unit
A: High conductance calcium-activated potassium channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,84324
Polymers120,3081
Non-polymers11,53523
Water0
1
A: High conductance calcium-activated potassium channel
hetero molecules

A: High conductance calcium-activated potassium channel
hetero molecules

A: High conductance calcium-activated potassium channel
hetero molecules

A: High conductance calcium-activated potassium channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)527,37296
Polymers481,2324
Non-polymers46,14092
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
SymmetryPoint symmetry: (Schoenflies symbol: C (n fold cyclic))

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Components

#1: Protein High conductance calcium-activated potassium channel


Mass: 120308.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aplysia californica (California sea hare)
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5QJC5
#2: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Chemical
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL / Phosphatidylglycerol


Mass: 749.007 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM

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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+ bound aplysia Slo1 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Aplysia californica (California sea hare)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Cell: High Five / Plasmid: pFastBac
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
2320 mMKClKCl1
310 mMCaCl2CaCl21
410 mMMgCl2MgCl21
520 mMDTTDTT1
62 mMTCEPTCEP1
70.025 %DDMDDM1
80.005 %CHSCHS1
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 293 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 magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 1.8 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: 2000
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: REFMAC / Version: 5.8.0088 / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10FREALIGNfinal Euler assignment
11RELIONclassification
12FREALIGN3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 233000
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115000 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 3.5→3.5 Å / Cor.coef. Fo:Fc: 0.85 / SU B: 39.858 / SU ML: 0.48 / ESU R: 1.568
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.328 --
obs0.328 32498 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 291.609 Å2
Baniso -1Baniso -2Baniso -3
1--0.24 Å20 Å20 Å2
2---0.24 Å20 Å2
3---0.49 Å2
Refinement stepCycle: 1 / Total: 7212
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0197356
ELECTRON MICROSCOPYr_bond_other_d0.0010.027068
ELECTRON MICROSCOPYr_angle_refined_deg0.9491.9699934
ELECTRON MICROSCOPYr_angle_other_deg0.805316206
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.2665883
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.15323.653323
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.644151178
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.3871540
ELECTRON MICROSCOPYr_chiral_restr0.0510.21111
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.028139
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021740
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.68230.4593553
ELECTRON MICROSCOPYr_mcbond_other3.67830.4593552
ELECTRON MICROSCOPYr_mcangle_it6.4145.6864429
ELECTRON MICROSCOPYr_mcangle_other6.4145.6874430
ELECTRON MICROSCOPYr_scbond_it3.76631.1723803
ELECTRON MICROSCOPYr_scbond_other3.76631.1723804
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other6.89246.5715506
ELECTRON MICROSCOPYr_long_range_B_refined10.6097953
ELECTRON MICROSCOPYr_long_range_B_other10.6097954
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.789 2393 -
Rfree-0 -
obs--100 %

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