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- EMDB-9025: The voltage-activated Kv1.2-2.1 paddle chimera channel in lipid n... -

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Entry
Database: EMDB / ID: EMD-9025
TitleThe voltage-activated Kv1.2-2.1 paddle chimera channel in lipid nanodiscs, cytosolic domain
Map dataKv1.2-2.1 paddle chimera in lipid nanodiscs, cytoplasmic domain
Sample
  • Complex: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1 paddle chimera in lipid nanodiscs
    • Protein or peptide: Voltage-gated potassium channel subunit beta-2
    • Protein or peptide: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: water
KeywordsMEMBRANE PROTEIN / transport protein / potassium channel / lipid nanodisc
Function / homology
Function and homology information


optic nerve structural organization / pinceau fiber / regulation of action potential / methylglyoxal reductase (NADPH) (acetol producing) activity / Voltage gated Potassium channels / voltage-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / potassium channel complex / NADPH oxidation / paranodal junction / regulation of circadian sleep/wake cycle, non-REM sleep ...optic nerve structural organization / pinceau fiber / regulation of action potential / methylglyoxal reductase (NADPH) (acetol producing) activity / Voltage gated Potassium channels / voltage-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / potassium channel complex / NADPH oxidation / paranodal junction / regulation of circadian sleep/wake cycle, non-REM sleep / potassium ion export across plasma membrane / axon initial segment / regulation of protein localization to cell surface / corpus callosum development / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / aldo-keto reductase (NADPH) activity / delayed rectifier potassium channel activity / outward rectifier potassium channel activity / juxtaparanode region of axon / Oxidoreductases; Acting on the CH-OH group of donors; With NAD+ or NADP+ as acceptor / regulation of potassium ion transmembrane transport / myoblast differentiation / Neutrophil degranulation / neuromuscular process / optic nerve development / action potential / regulation of dopamine secretion / neuronal cell body membrane / kinesin binding / lamellipodium membrane / calyx of Held / voltage-gated potassium channel activity / neuronal action potential / potassium channel regulator activity / hematopoietic progenitor cell differentiation / voltage-gated potassium channel complex / axon terminus / potassium ion transmembrane transport / sensory perception of pain / protein localization to plasma membrane / postsynaptic density membrane / potassium ion transport / protein homooligomerization / cerebral cortex development / cytoplasmic side of plasma membrane / lamellipodium / presynaptic membrane / perikaryon / postsynaptic membrane / transmembrane transporter binding / postsynaptic density / cytoskeleton / endosome / neuron projection / protein heterodimerization activity / axon / neuronal cell body / glutamatergic synapse / dendrite / endoplasmic reticulum membrane / protein-containing complex binding / membrane / plasma membrane / cytosol
Similarity search - Function
Potassium channel, voltage dependent, Kv2.2 / Potassium channel, voltage dependent, Kv2 / Kv2 voltage-gated K+ channel / Potassium channel, voltage dependent, Kv1.2 / 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 / Kv2 voltage-gated K+ channel / Potassium channel, voltage dependent, Kv1.2 / 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 / 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
Similarity search - Domain/homology
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMatthies D / Bae C
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, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseAug 22, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ebl
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9025.png

Downloads & links

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Map

FileDownload / File: emd_9025.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKv1.2-2.1 paddle chimera in lipid nanodiscs, cytoplasmic domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å		0.84 Å/pix.
x 384 pix.
= 320.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.12877041 - 0.18350628
Average (Standard dev.)-0.000004978093 (±0.0043570735)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8350.8350.835
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z320.640320.640320.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-36
NX/NY/NZ528549
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1290.184-0.000

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Supplemental data

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Mask #1

Fileemd_9025_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1...

EntireName: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1 paddle chimera in lipid nanodiscs
Components
  • Complex: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1 paddle chimera in lipid nanodiscs
    • Protein or peptide: Voltage-gated potassium channel subunit beta-2
    • Protein or peptide: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: water

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Supramolecule #1: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1...

SupramoleculeName: Cytosolic domain of voltage-activated potassium channel Kv1.2-2.1 paddle chimera in lipid nanodiscs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 385 KDa

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Macromolecule #1: Voltage-gated potassium channel subunit beta-2

MacromoleculeName: Voltage-gated potassium channel subunit beta-2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 37.339059 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MVQFYRNLGK SGLRVSCLGL GTWVTFGGQI TDEMAEHLMT LAYDNGINLF DTAEVYAAGK AEVVLGNIIK KKGWRRSSLV ITTKIFWGG KAETERGLSR KHIIEGLKAS LERLQLEYVD VVFANRPDPN TPMEETVRAM THVINQGMAM YWGTSRWSSM E IMEAYSVA ...String:
MVQFYRNLGK SGLRVSCLGL GTWVTFGGQI TDEMAEHLMT LAYDNGINLF DTAEVYAAGK AEVVLGNIIK KKGWRRSSLV ITTKIFWGG KAETERGLSR KHIIEGLKAS LERLQLEYVD VVFANRPDPN TPMEETVRAM THVINQGMAM YWGTSRWSSM E IMEAYSVA RQFNLIPPIC EQAEYHMFQR EKVEVQLPEL FHKIGVGAMT WSPLACGIVS GKYDSGIPPY SRASLKGYQW LK DKILSEE GRRQQAKLKE LQAIAERLGC TLPQLAIAWC LRNEGVSSVL LGASNAEQLM ENIGAIQVLP KLSSSIVHEI DSI LGNKPY SKKDYRS

UniProtKB: Voltage-gated potassium channel subunit beta-2

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Macromolecule #2: Potassium voltage-gated channel subfamily A member 2,Potassium vo...

MacromoleculeName: Potassium voltage-gated channel subfamily A member 2,Potassium voltage-gated channel subfamily B member 2 chimera
type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 58.905828 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MAHHHHHHHH ENLYFQGSMT VATGDPVDEA AAHPGHPQDT YDPEADHECC ERVVINISGL RFETQLKTLA QFPETLLGDP KKRMRYFDP LRNEYFFDRN RPSFDAILYY YQSGGRLRRP VNVPLDIFSE EIRFYELGEE AMEMFREDEG YIKEEERPLP E NEFQRQVW ...String:
MAHHHHHHHH ENLYFQGSMT VATGDPVDEA AAHPGHPQDT YDPEADHECC ERVVINISGL RFETQLKTLA QFPETLLGDP KKRMRYFDP LRNEYFFDRN RPSFDAILYY YQSGGRLRRP VNVPLDIFSE EIRFYELGEE AMEMFREDEG YIKEEERPLP E NEFQRQVW LLFEYPESSG PARIIAIVSV MVILISIVSF CLETLPIFRD ENEDMHGGGV TFHTYSQSTI GYQQSTSFTD PF FIVETLC IIWFSFEFLV RFFACPSKAG FFTNIMNIID IVAIIPYYVT IFLTESNKSV LQFQNVRRVV QIFRIMRILR IFK LSRHSK GLQILGQTLK ASMRELGLLI FFLFIGVILF SSAVYFAEAD ERDSQFPSIP DAFWWAVVSM TTVGYGDMVP TTIG GKIVG SLCAIAGVLT IALPVPVIVS NFNYFYHRET EGEEQAQYLQ VTSCPKIPSS PDLKKSRSAS TISKSDYMEI QEGVN NSNE DFREENLKTA NCTLANTNYV NITKMLTDV

UniProtKB: Potassium voltage-gated channel subfamily A member 2, Potassium voltage-gated channel subfamily B member 2, Potassium voltage-gated channel subfamily A member 2

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Macromolecule #3: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 128 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES-KOHHEPES-KOH
150.0 mMKClpotassium chloride
2.0 mMTCEPTCEP
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 88 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
Details: A 3 microliter sample was applied to a plasma-cleaned grid and blotted for 10 seconds..
DetailsCytosolic domain of Kv1.2-2.1 paddle chimera in lipid nanodiscs obtained after signal subtraction in RELION 2.1

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 3085 / Average exposure time: 15.2 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 281021
Startup modelType of model: OTHER
Details: An initial model was created from negatively stained single particle electron microscopy.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 57384
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 2.1)

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Atomic model buiding 1

Initial modelPDB ID:

2r9r


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 84
Output model

PDB-6ebl:
The voltage-activated Kv1.2-2.1 paddle chimera channel in lipid nanodiscs, cytosolic domain

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