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- PDB-5u70: Chicken Slo2.2 in an open conformation vitrified in the presence ... -

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Basic information

Entry
Database: PDB / ID: 5u70
TitleChicken Slo2.2 in an open conformation vitrified in the presence of 300 mM NaCl
ComponentsPotassium channel subfamily T member 1
KeywordsTRANSPORT PROTEIN / Ion channel / potassium channel
Function / homology
Function and homology information


intracellular sodium-activated potassium channel activity / outward rectifier potassium channel activity / potassium ion transmembrane transport / plasma membrane
Similarity search - Function
Calcium-activated potassium channel BK, alpha subunit / : / Calcium-activated BK potassium channel alpha subunit / Calcium-activated potassium channel slowpoke-like RCK domain / RCK N-terminal domain profile. / Potassium channel domain / Ion channel
Similarity search - Domain/homology
Potassium channel subfamily T member 1
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsHite, R.K. / MacKinnon, R.
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: Cell / Year: 2017
Title: Structural Titration of Slo2.2, a Na-Dependent K Channel.
Authors: Richard K Hite / Roderick MacKinnon /
Abstract: The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of ...The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na-activated K channel, as a function of the Na concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na-dependent manner, without evidence of Na-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.
History
DepositionDec 9, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.details / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software / Item: _em_sample_support.grid_type / _em_software.name
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 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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Structure viewerMolecule:
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Assembly

Deposited unit
A: Potassium channel subfamily T member 1


Theoretical massNumber of molelcules
Total (without water)137,4101
Polymers137,4101
Non-polymers00
Water00
1
A: Potassium channel subfamily T member 1

A: Potassium channel subfamily T member 1

A: Potassium channel subfamily T member 1

A: Potassium channel subfamily T member 1


Theoretical massNumber of molelcules
Total (without water)549,6394
Polymers549,6394
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C4 (4 fold cyclic))

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Components

#1: Protein Potassium channel subfamily T member 1 / Slo2.2


Mass: 137409.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: KCNT1, SLACK / Plasmid: pFastBac / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8QFV0

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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: Chicken Slo2.2 in open conformation / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: pFastBac
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2300 mMpotassium chlorideKCl1
3300 mMsodium chlorideNaCl1
41.5 mMdodecyl maltoside1
50.075 mg/ml1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine1
60.025 mg/ml1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol1
SpecimenConc.: 6 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: 88 % / Chamber temperature: 298 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: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 59 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: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: REFMAC / Version: 5.8.0088 / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4CTFFIND44.0.16CTF correction
9PHENIXmodel refinementphenix.real_space_refine
10REFMACmodel refinement
11RELION2initial Euler assignment
12FREALIGNfinal Euler assignment
13RELION2classification
14FREALIGN3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 160000 / Details: Manual particle selection in RELION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130000 / Algorithm: FOURIER SPACE / Num. of class averages: 8 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementResolution: 3.76→3.76 Å / Cor.coef. Fo:Fc: 0.889 / SU B: 46.11 / SU ML: 0.499 / ESU R: 1.395
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.33324 --
obs0.33324 34823 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: 384.843 Å2
Baniso -1Baniso -2Baniso -3
1--0.53 Å20 Å20 Å2
2---0.53 Å20 Å2
3---1.05 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0197453
ELECTRON MICROSCOPYr_bond_other_d0.0010.027187
ELECTRON MICROSCOPYr_angle_refined_deg0.911.96510093
ELECTRON MICROSCOPYr_angle_other_deg0.794316497
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.0285897
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.96423.253332
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.276151318
ELECTRON MICROSCOPYr_dihedral_angle_4_deg8.8561550
ELECTRON MICROSCOPYr_chiral_restr0.050.21138
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0218262
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021770
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_it5.438.4153602
ELECTRON MICROSCOPYr_mcbond_other5.39538.4153601
ELECTRON MICROSCOPYr_mcangle_it9.09557.6324493
ELECTRON MICROSCOPYr_mcangle_other9.09557.6324494
ELECTRON MICROSCOPYr_scbond_it6.09139.4913851
ELECTRON MICROSCOPYr_scbond_other6.0939.4913852
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other10.95858.8835601
ELECTRON MICROSCOPYr_long_range_B_refined15.4978300
ELECTRON MICROSCOPYr_long_range_B_other15.4978301
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.842 2560 -
Rfree-0 -
obs--100 %

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