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- EMDB-20294: Single particle cryo-EM structure of the voltage-gated K+ channel... -

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Basic information

Entry
Database: EMDB / ID: EMD-20294
TitleSingle particle cryo-EM structure of the voltage-gated K+ channel Eag1 3-13 deletion mutant bound to calmodulin (conformation 2)
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


VEGFR2 mediated cell proliferation / RAF activation / RAF/MAP kinase cascade / CaMK IV-mediated phosphorylation of CREB / Calmodulin induced events / Cam-PDE 1 activation / CaM pathway / Platelet degranulation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases activate PAKs ...VEGFR2 mediated cell proliferation / RAF activation / RAF/MAP kinase cascade / CaMK IV-mediated phosphorylation of CREB / Calmodulin induced events / Cam-PDE 1 activation / CaM pathway / Platelet degranulation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases activate PAKs / Signaling by moderate kinase activity BRAF mutants / Phase 0 - rapid depolarisation / Ion homeostasis / CLEC7A (Dectin-1) induces NFAT activation / RHO GTPases activate IQGAPs / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Signaling by RAS mutants / Activation of AMPK downstream of NMDARs / Loss of phosphorylation of MECP2 at T308 / Regulation of MECP2 expression and activity / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Ion transport by P-type ATPases / Negative regulation of NMDA receptor-mediated neuronal transmission / Activation of RAC1 downstream of NMDARs / Long-term potentiation / Unblocking of NMDA receptors, glutamate binding and activation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Ras activation upon Ca2+ influx through NMDA receptor / Smooth Muscle Contraction / Voltage gated Potassium channels / Activation of Ca-permeable Kainate Receptor / Uptake and function of anthrax toxins / VEGFR2 mediated vascular permeability / Extra-nuclear estrogen signaling / CREB1 phosphorylation through the activation of Adenylate Cyclase / Sodium/Calcium exchangers / Reduction of cytosolic Ca++ levels / Signaling by BRAF and RAF fusions / PKA activation / DARPP-32 events / Synthesis of IP3 and IP4 in the cytosol / Paradoxical activation of RAF signaling by kinase inactive BRAF / Glycogen breakdown (glycogenolysis) / Calcineurin activates NFAT / eNOS activation / Transcriptional activation of mitochondrial biogenesis / Inactivation, recovery and regulation of the phototransduction cascade / Stimuli-sensing channels / FCERI mediated Ca+2 mobilization / Ca2+ pathway / Protein methylation / potassium channel complex / delayed rectifier potassium channel activity / phosphatidylinositol bisphosphate binding / startle response / nuclear inner membrane / axolemma / 14-3-3 protein binding / glycogen catabolic process / go:0075206: / N-terminal myristoylation domain binding / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / potassium ion transmembrane transport / inositol phosphate metabolic process / protein phosphatase activator activity / positive regulation of ryanodine-sensitive calcium-release channel activity / catalytic complex / regulation of rhodopsin mediated signaling pathway / positive regulation of cyclic-nucleotide phosphodiesterase activity / adenylate cyclase activator activity / voltage-gated potassium channel activity / adenylate cyclase binding / detection of calcium ion / voltage-gated potassium channel complex / regulation of ion transmembrane transport / negative regulation of ryanodine-sensitive calcium-release channel activity / Wnt signaling pathway, calcium modulating pathway / positive regulation of protein dephosphorylation / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / regulation of cardiac muscle contraction / positive regulation of phosphoprotein phosphatase activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / protein serine/threonine kinase activator activity / calcium channel complex / regulation of membrane potential / titin binding / substantia nigra development / cofactor metabolic process / regulation of ryanodine-sensitive calcium-release channel activity / sarcomere / ion transmembrane transport / regulation of heart rate / positive regulation of protein serine/threonine kinase activity / cellular response to calcium ion / positive regulation of protein autophosphorylation / muscle contraction / phosphatidylinositol-mediated signaling / positive regulation of peptidyl-threonine phosphorylation
PAC domain profile. / Ion transport protein / PAS domain / EF-hand domain pair / EF-hand calcium-binding domain. / cAMP/cGMP binding motif profile. / PAS repeat profile. / EF-hand calcium-binding domain profile. / Cyclic nucleotide-binding domain / Calmodulin ...PAC domain profile. / Ion transport protein / PAS domain / EF-hand domain pair / EF-hand calcium-binding domain. / cAMP/cGMP binding motif profile. / PAS repeat profile. / EF-hand calcium-binding domain profile. / Cyclic nucleotide-binding domain / Calmodulin / EF-hand domain pair / Cyclic nucleotide-binding domain / PAS-associated, C-terminal / PAC motif / EF-hand domain / Potassium channel, voltage-dependent, EAG/ELK/ERG / Potassium channel, voltage-dependent, EAG / Ion transport domain / RmlC-like jelly roll fold / PAS domain superfamily / EF-Hand 1, calcium-binding site / Cyclic nucleotide-binding-like / Voltage-dependent channel domain superfamily / Potassium voltage-gated channel subfamily H member 1 / PAS domain
Calmodulin-1 / Potassium voltage-gated channel subfamily H member 1
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsWhicher JR / MacKinnon R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM43949 United States
Damon Runyon Cancer Research FoundationDRG-2212-15 United States
Howard Hughes Medical Institute 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, ...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: 6pbx

SummaryFull reportAbout validation report
History
DepositionJun 14, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 18, 2019-
UpdateSep 25, 2019-
Current statusSep 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6pbx
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6pbx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20294.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: 0.0124 / Movie #1: 0.0124
Minimum - Maximum-0.027176592 - 0.052881088
Average (Standard dev.)0.000019980069 (±0.002193549)
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-0.0270.0530.000

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Supplemental data

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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: 54530
3D reconstructionSoftware: RELION / Resolution: 4 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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