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- EMDB-20295: Single particle cryo-EM structure of the voltage-gated K+ channel... -

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Basic information

Entry
Database: EMDB / ID: EMD-20295
TitleSingle particle cryo-EM structure of the voltage-gated K+ channel Eag1 3-13 deletion mutant bound to calmodulin (conformation 1)
Map data
SampleVoltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
  • Voltage-gated potassium channel Eag1 3-13 deletion mutant
  • calmodulin
  • Potassium voltage-gated channel subfamily H member 1
  • Calmodulin-1
Function / homology
Function and homology information


voltage-gated ion channel activity involved in regulation of presynaptic membrane potential / regulation of presynaptic cytosolic calcium ion concentration / potassium channel complex / delayed rectifier potassium channel activity / phosphatidylinositol bisphosphate binding / parallel fiber to Purkinje cell synapse / nuclear inner membrane / startle response / axolemma / 14-3-3 protein binding ...voltage-gated ion channel activity involved in regulation of presynaptic membrane potential / regulation of presynaptic cytosolic calcium ion concentration / potassium channel complex / delayed rectifier potassium channel activity / phosphatidylinositol bisphosphate binding / parallel fiber to Purkinje cell synapse / nuclear inner membrane / startle response / axolemma / 14-3-3 protein binding / glycogen catabolic process / N-terminal myristoylation domain binding / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / inositol phosphate metabolic process / protein phosphatase activator activity / positive regulation of ryanodine-sensitive calcium-release channel activity / potassium ion transmembrane transport / regulation of rhodopsin mediated signaling pathway / detection of calcium ion / catalytic complex / voltage-gated potassium channel complex / adenylate cyclase binding / positive regulation of cyclic-nucleotide phosphodiesterase activity / adenylate cyclase activator activity / voltage-gated potassium channel activity / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / regulation of ion transmembrane transport / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / positive regulation of phosphoprotein phosphatase activity / Wnt signaling pathway, calcium modulating pathway / positive regulation of protein dephosphorylation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / go:0051186: / titin binding / substantia nigra development / calcium channel complex / sarcomere / regulation of ryanodine-sensitive calcium-release channel activity / regulation of heart rate / protein serine/threonine kinase activator activity / positive regulation of protein autophosphorylation / cellular response to calcium ion / enzyme regulator activity / postsynaptic density membrane / phosphatidylinositol-mediated signaling / ion transmembrane transport / regulation of synaptic vesicle exocytosis / spindle microtubule / regulation of membrane potential / positive regulation of peptidyl-threonine phosphorylation / integral component of presynaptic membrane / positive regulation of protein serine/threonine kinase activity / regulation of nitric-oxide synthase activity / regulation of cytokinesis / presynaptic membrane / calcium-mediated signaling / muscle contraction / response to calcium ion / microtubule cytoskeleton organization / spindle pole / platelet degranulation / Fc-epsilon receptor signaling pathway / early endosome membrane / disordered domain specific binding / regulation of cell population proliferation / ion channel binding / perikaryon / vesicle / calmodulin binding / MAPK cascade / centrosome / G protein-coupled receptor signaling pathway / synapse / axon / protein domain specific binding / neuronal cell body / dendrite / protein-containing complex binding / calcium ion binding / protein kinase binding / viral process / perinuclear region of cytoplasm / cell surface / integral component of plasma membrane / protein-containing complex / extracellular region / nucleoplasm / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm
PAS domain / PAS domain superfamily / Ion transport domain / EF-hand domain pair / EF-hand domain / RmlC-like jelly roll fold / EF-Hand 1, calcium-binding site / Cyclic nucleotide-binding-like / Voltage-dependent channel domain superfamily / Potassium channel, voltage-dependent, EAG/ELK/ERG ...PAS domain / PAS domain superfamily / Ion transport domain / EF-hand domain pair / EF-hand domain / RmlC-like jelly roll fold / EF-Hand 1, calcium-binding site / Cyclic nucleotide-binding-like / Voltage-dependent channel domain superfamily / Potassium channel, voltage-dependent, EAG/ELK/ERG / Potassium voltage-gated channel subfamily H member 1 / PAC motif / PAS-associated, C-terminal / Calmodulin / Cyclic nucleotide-binding domain / Potassium channel, voltage-dependent, EAG
Calmodulin-1 / Potassium voltage-gated channel subfamily H member 1
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsWhicher JR / MacKinnon R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
Damon Runyon Cancer Research FoundationDRG-2212-15 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2019
Title: Regulation of Eag1 gating by its intracellular domains.
Authors: Jonathan R Whicher / Roderick MacKinnon /
Abstract: Voltage-gated potassium channels (Ks) are gated by transmembrane voltage sensors (VS) that move in response to changes in membrane voltage. K10.1 or Eag1 also has three intracellular domains: PAS, C- ...Voltage-gated potassium channels (Ks) are gated by transmembrane voltage sensors (VS) that move in response to changes in membrane voltage. K10.1 or Eag1 also has three intracellular domains: PAS, C-linker, and CNBHD. We demonstrate that the Eag1 intracellular domains are not required for voltage-dependent gating but likely interact with the VS to modulate gating. We identified specific interactions between the PAS, CNBHD, and VS that modulate voltage-dependent gating and provide evidence that VS movement destabilizes these interactions to promote channel opening. Additionally, mutation of these interactions renders Eag1 insensitive to calmodulin inhibition. The structure of the calmodulin insensitive mutant in a pre-open conformation suggests that channel opening may occur through a rotation of the intracellular domains and calmodulin may prevent this rotation by stabilizing interactions between the VS and intracellular domains. Intracellular domains likely play a similar modulatory role in voltage-dependent gating of the related K11-12 channels.
Validation ReportPDB-ID: 6pby

SummaryFull reportAbout validation report
History
DepositionJun 14, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 18, 2019-
UpdateNov 20, 2019-
Current statusNov 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pby
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20295.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 320 pix.
= 320. Å
1 Å/pix.
x 320 pix.
= 320. Å
1 Å/pix.
x 320 pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0156 / Movie #1: 0.0156
Minimum - Maximum-0.05044838 - 0.09725
Average (Standard dev.)0.0000418478 (±0.0029395586)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0500.0970.000

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Supplemental data

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Additional map: Unsharpened map for Eag1 3-13/CaM conformation 1

Fileemd_20295_additional.map
AnnotationUnsharpened map for Eag1 3-13/CaM conformation 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Voltage-gated potassium channel Eag1 3-13 deletion mutant bound t...

EntireName: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
Number of components: 5

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Component #1: protein, Voltage-gated potassium channel Eag1 3-13 deletion mutan...

ProteinName: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
Recombinant expression: No

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Component #2: protein, Voltage-gated potassium channel Eag1 3-13 deletion mutant

ProteinName: Voltage-gated potassium channel Eag1 3-13 deletion mutant
Recombinant expression: No
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, calmodulin

ProteinName: calmodulin / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #4: protein, Potassium voltage-gated channel subfamily H member 1

ProteinName: Potassium voltage-gated channel subfamily H member 1 / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 96.26143 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Homo sapiens (human)

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Component #5: protein, Calmodulin-1

ProteinName: Calmodulin-1 / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 16.852545 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.6 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C4 (4 fold cyclic) / Number of projections: 43137
3D reconstructionSoftware: RELION / Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 5K7L
Output model

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