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- PDB-6ebl: The voltage-activated Kv1.2-2.1 paddle chimera channel in lipid n... -

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Basic information

Entry
Database: PDB / ID: 6ebl
TitleThe voltage-activated Kv1.2-2.1 paddle chimera channel in lipid nanodiscs, cytosolic domain
Components
  • Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
  • Voltage-gated potassium channel subunit beta-2
KeywordsMEMBRANE PROTEIN / transport protein / potassium channel / lipid nanodisc
Function / homology
Function and homology information


optic nerve structural organization / pinceau fiber / regulation of action potential / Voltage gated Potassium channels / voltage-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / potassium channel complex / NADPH oxidation / paranodal junction / potassium ion export across plasma membrane / regulation of circadian sleep/wake cycle, non-REM sleep ...optic nerve structural organization / pinceau fiber / regulation of action potential / Voltage gated Potassium channels / voltage-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / potassium channel complex / NADPH oxidation / paranodal junction / potassium ion export across plasma membrane / regulation of circadian sleep/wake cycle, non-REM sleep / axon initial segment / regulation of protein localization to cell surface / delayed rectifier potassium channel activity / corpus callosum development / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / aldo-keto reductase (NADPH) activity / outward rectifier potassium channel activity / juxtaparanode region of axon / optic nerve development / regulation of potassium ion transmembrane transport / Oxidoreductases; Acting on the CH-OH group of donors; With NAD+ or NADP+ as acceptor / myoblast differentiation / Neutrophil degranulation / neuromuscular process / neuronal cell body membrane / regulation of dopamine secretion / voltage-gated potassium channel activity / lamellipodium membrane / kinesin binding / neuronal action potential / calyx of Held / potassium channel regulator activity / hematopoietic progenitor cell differentiation / voltage-gated potassium channel complex / axon terminus / sensory perception of pain / potassium ion transmembrane transport / extrinsic component of cytoplasmic side of plasma membrane / protein localization to plasma membrane / postsynaptic density membrane / protein homooligomerization / potassium ion transport / cytoplasmic side of plasma membrane / cerebral cortex development / lamellipodium / presynaptic membrane / perikaryon / postsynaptic membrane / transmembrane transporter binding / postsynaptic density / cytoskeleton / endosome / neuron projection / protein heterodimerization activity / axon / dendrite / neuronal cell body / glutamatergic synapse / protein-containing complex binding / endoplasmic reticulum membrane / membrane / plasma membrane / cytosol
Similarity search - Function
Potassium channel, voltage dependent, Kv2.2 / Potassium channel, voltage dependent, Kv2 / Potassium channel, voltage dependent, Kv1.2 / Kv2 voltage-gated K+ channel / Potassium channel, voltage-dependent, beta subunit, KCNAB2 / Potassium channel, voltage-dependent, beta subunit, KCNAB / Potassium channel, voltage-dependent, beta subunit, KCNAB-related / Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain ...Potassium channel, voltage dependent, Kv2.2 / Potassium channel, voltage dependent, Kv2 / Potassium channel, voltage dependent, Kv1.2 / Kv2 voltage-gated K+ channel / Potassium channel, voltage-dependent, beta subunit, KCNAB2 / Potassium channel, voltage-dependent, beta subunit, KCNAB / Potassium channel, voltage-dependent, beta subunit, KCNAB-related / Potassium channel, voltage dependent, Kv1 / Potassium channel, voltage dependent, Kv / Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Potassium Channel Kv1.1; Chain A / Potassium Channel Kv1.1; Chain A / NADP-dependent oxidoreductase domain / NADP-dependent oxidoreductase domain / Aldo/keto reductase family / NADP-dependent oxidoreductase domain superfamily / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / Voltage-dependent channel domain superfamily / SKP1/BTB/POZ domain superfamily / Ion transport domain / Ion transport protein / TIM Barrel / Alpha-Beta Barrel / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Voltage-gated potassium channel subunit beta-2 / Potassium voltage-gated channel subfamily A member 2 / Potassium voltage-gated channel subfamily B member 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsMatthies, D. / Bae, C. / Fox, T. / Bartesaghi, A. / Subramaniam, S. / Swartz, K.J.
CitationJournal: Elife / Year: 2018
Title: Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs.
Authors: Doreen Matthies / Chanhyung Bae / Gilman Es Toombes / Tara Fox / Alberto Bartesaghi / Sriram Subramaniam / Kenton Jon Swartz /
Abstract: Voltage-activated potassium (Kv) channels open to conduct K ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X- ...Voltage-activated potassium (Kv) channels open to conduct K ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. Here we report the structure of the Kv1.2-2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy. At a resolution of ~3 Å for the cytosolic domain and ~4 Å for the transmembrane domain, the structure determined in nanodiscs is similar to the previously determined X-ray structure. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.
History
DepositionAug 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 22, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 13, 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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Assembly

Deposited unit
A: Voltage-gated potassium channel subunit beta-2
B: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
C: Voltage-gated potassium channel subunit beta-2
D: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
E: Voltage-gated potassium channel subunit beta-2
F: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
G: Voltage-gated potassium channel subunit beta-2
H: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)387,95312
Polymers384,9808
Non-polymers2,9744
Water2,306128
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis, microscopy, negative staining single particle electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21870 Å2
ΔGint-38 kcal/mol
Surface area66700 Å2

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Components

#1: Protein
Voltage-gated potassium channel subunit beta-2 / / K(+) channel subunit beta-2 / Kv-beta-2


Mass: 37339.059 Da / Num. of mol.: 4 / Mutation: cytosolic domain (UNP residues 37-367)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Kcnab2, Ckbeta2, Kcnb3 / Production host: Komagataella pastoris (fungus) / References: UniProt: P62483
#2: Protein
Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera / RAK / RBK2 / RCK5 / Voltage-gated potassium channel subunit Kv1.2 / CDRK / Voltage-gated potassium ...RAK / RBK2 / RCK5 / Voltage-gated potassium channel subunit Kv1.2 / CDRK / Voltage-gated potassium channel subunit Kv2.2 / RAK / RBK2 / RCK5 / Voltage-gated potassium channel subunit Kv1.2


Mass: 58905.828 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Kcna2, Kcnb2 / Production host: Komagataella pastoris (fungus) / References: UniProt: P63142, UniProt: Q63099
#3: Chemical
ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 128 / Source method: isolated from a natural source / Formula: H2O

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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: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1 paddle chimera in lipid nanodiscs
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.385 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Komagataella pastoris (fungus) / Plasmid: pPICZ
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOHHEPES-KOH1
2150 mMpotassium chlorideKCl1
32 mMTCEPTCEP1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Cytosolic domain of Kv1.2-2.1 paddle chimera in lipid nanodiscs obtained after signal subtraction in RELION 2.1
Specimen supportGrid material: GOLD / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 88 % / Chamber temperature: 277.15 K
Details: A 3 microliter sample was applied to a plasma-cleaned grid and blotted for 10 seconds.

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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: 29000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 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: 15.2 sec. / Electron dose: 40 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: 3085
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 38 / Used frames/image: 2-20

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Processing

EM software
IDNameVersionCategory
2Latitude3.21.1253image acquisition
7UCSF Chimeramodel fitting
8Cootmodel fitting
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
14Cootmodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 281021
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57384 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 84 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 2R9R

2r9r


Accession code: 2R9R / Source name: PDB / Type: experimental model

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