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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/homologyCalcium-activated potassium channel Slo-1 / large conductance calcium-activated potassium channel activity / 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 / integral component of membrane / metal ion binding / High conductance calcium-activated potassium channel
Function and homology information
Specimen sourceAplysia californica / invertebrata / California sea hare / image: Aplysia kurodai
MethodElectron microscopy (3.5 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsMacKinnon, R. / Tao, X. / Hite, R.K.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 3, 2016 / Release: Dec 14, 2016
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 14, 2016Structure modelrepositoryInitial release
1.1Jan 11, 2017Structure modelDatabase references
1.2Jan 18, 2017Structure modelDatabase references
1.3Sep 27, 2017Structure modelAuthor supporting evidence / Data collectionem_software / pdbx_audit_support_em_software.name / _pdbx_audit_support.funding_organization

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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
Polyers120,3081
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
Polyers481,2324
Non-polymers46,14092
Water0
TypeNameSymmetry operationNumber
point symmetry operation4

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Components

#1: Protein/peptide High conductance calcium-activated potassium channel


Mass: 120308.000 Da / Num. of mol.: 1
Source: (gene. exp.) Aplysia californica / invertebrata / California sea hare / image: Aplysia kurodai
Production host: Trichoplusia ni / References: UniProt:Q5QJC5
#2: Chemical
ChemComp-K / POTASSIUM ION


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


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


Mass: 40.078 Da / Num. of mol.: 2 / Formula: Ca / : Calcium
#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


Mass: 749.007 Da / Num. of mol.: 15 / Formula: C40H77O10P / : Phosphatidylglycerol / Comment: phospholipid *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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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 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Aplysia californica
Source (recombinant)Cell: High Five / Organism: Trichoplusia ni / Plasmid: pFastBac
Buffer solutionpH: 8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mMTrisTris1
2320mMKClKCl1
310mMCaCl2CaCl21
410mMMgCl2MgCl21
520mMDTTDTT1
62mMTCEPTCEP1
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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 293 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 / 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) / Number of grids imaged: 1 / Number of real images: 2000
Image scansSampling size: 5 microns / Dimension width: 7420 / Dimension 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
12FREALIGNRECONSTRUCTION
13REFMACMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 233000
SymmetryPoint symmetry: C4
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 115000 / Symmetry type: POINT
Atomic model buildingOverall b value: 100 / Ref protocol: AB INITIO MODEL / Ref space: RECIPROCAL
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcDetailsR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLOverall ESU RSolvent ion probe radiiSolvent shrinkage radiiSolvent vdw probe radiiStereochemistry target valuesSolvent model details
1291.609-0.240.000.00-0.240.000.490.850HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.328000.328003.40141.0032498100.0039.8580.4801.5680.800.801.20MAXIMUM LIKELIHOOD WITH PHASESMASK
ELECTRON MICROSCOPY
Number of atoms included #1Total: 7212
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0197356
ELECTRON MICROSCOPYr_bond_other_d0.0010.0207068
ELECTRON MICROSCOPYr_angle_refined_deg0.9491.9699934
ELECTRON MICROSCOPYr_angle_other_deg0.8053.00016206
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.2665.000883
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.15323.653323
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.64415.0001178
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.38715.00040
ELECTRON MICROSCOPYr_chiral_restr0.0510.2001111
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0208139
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0201740
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_it6.41045.6864429
ELECTRON MICROSCOPYr_mcangle_other6.41045.6874430
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
Refine LS shellHighest resolution: 3.4 Å / R factor R work: 0.789 / Lowest resolution: 3.488 Å / Number reflection R free: 0 / Number reflection R work: 2393 / Total number of bins used: 20 / Percent reflection obs: 1

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