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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 dataSharpened map for Eag1 3-13/CaM conformation 2
Sample
  • Complex: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
    • Complex: Voltage-gated potassium channel Eag1 3-13 deletion mutant
      • Protein or peptide: Potassium voltage-gated channel subfamily H member 1
    • Complex: calmodulin
      • Protein or peptide: Calmodulin-1
KeywordsVoltage-gated potassium channel / ion channel / calmodulin / TRANSPORT PROTEIN-CALCIUM BINDING PROTEIN complex
Function / homology
Function and homology information


Voltage gated Potassium channels / potassium channel complex / regulation of presynaptic cytosolic calcium ion concentration / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / delayed rectifier potassium channel activity / CaM pathway / parallel fiber to Purkinje cell synapse / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events ...Voltage gated Potassium channels / potassium channel complex / regulation of presynaptic cytosolic calcium ion concentration / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / delayed rectifier potassium channel activity / CaM pathway / parallel fiber to Purkinje cell synapse / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / nuclear inner membrane / Reduction of cytosolic Ca++ levels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Activation of Ca-permeable Kainate Receptor / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / negative regulation of high voltage-gated calcium channel activity / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / phosphatidylinositol bisphosphate binding / positive regulation of cyclic-nucleotide phosphodiesterase activity / organelle localization by membrane tethering / negative regulation of calcium ion export across plasma membrane / regulation of synaptic vesicle exocytosis / CLEC7A (Dectin-1) induces NFAT activation / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / Activation of RAC1 downstream of NMDARs / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / Negative regulation of NMDA receptor-mediated neuronal transmission / startle response / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Unblocking of NMDA receptors, glutamate binding and activation / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of phosphoprotein phosphatase activity / Ion transport by P-type ATPases / Long-term potentiation / Uptake and function of anthrax toxins / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / axolemma / negative regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / calcium channel inhibitor activity / RHO GTPases activate IQGAPs / cellular response to interferon-beta / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / eNOS activation / Activation of AMPK downstream of NMDARs / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / positive regulation of protein dephosphorylation / Ion homeostasis / regulation of calcium-mediated signaling / regulation of ryanodine-sensitive calcium-release channel activity / titin binding / monoatomic ion transmembrane transport / positive regulation of protein autophosphorylation / potassium ion transmembrane transport / voltage-gated potassium channel complex / sperm midpiece / 14-3-3 protein binding / calcium channel complex / cellular response to calcium ion / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / sarcomere / protein serine/threonine kinase activator activity / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / regulation of cytokinesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / positive regulation of peptidyl-threonine phosphorylation / VEGFR2 mediated cell proliferation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of membrane potential / postsynaptic density membrane / positive regulation of receptor signaling pathway via JAK-STAT / RAF activation / Transcriptional activation of mitochondrial biogenesis / positive regulation of protein serine/threonine kinase activity / spindle microtubule / Stimuli-sensing channels / spindle pole / cellular response to type II interferon / response to calcium ion
Similarity search - Function
Potassium channel, voltage-dependent, EAG / Potassium channel, voltage-dependent, EAG/ELK/ERG / PAS domain / PAS-associated, C-terminal / PAC domain profile. / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. ...Potassium channel, voltage-dependent, EAG / Potassium channel, voltage-dependent, EAG/ELK/ERG / PAS domain / PAS-associated, C-terminal / PAC domain profile. / PAC motif / Motif C-terminal to PAS motifs (likely to contribute to PAS structural domain) / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / PAS repeat profile. / RmlC-like jelly roll fold / PAS domain / PAS domain superfamily / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / Ion transport domain / Ion transport protein / EF-hand domain pair
Similarity search - Domain/homology
Calmodulin-1 / Potassium voltage-gated channel subfamily H member 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 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
Howard Hughes Medical Institute (HHMI) United States
Damon Runyon Cancer Research FoundationDRG-2212-15 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.
History
DepositionJun 14, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseSep 18, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing 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
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  • 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 modelsPDB-6pbx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20294.png

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)
AnnotationSharpened map for Eag1 3-13/CaM conformation 2
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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Additional map: Unsharpened map for Eag1 3-13/CaM conformation 2

Fileemd_20294_additional.map
AnnotationUnsharpened map for Eag1 3-13/CaM conformation 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
    • Complex: Voltage-gated potassium channel Eag1 3-13 deletion mutant
      • Protein or peptide: Potassium voltage-gated channel subfamily H member 1
    • Complex: calmodulin
      • Protein or peptide: Calmodulin-1

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Supramolecule #1: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound t...

SupramoleculeName: Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Voltage-gated potassium channel Eag1 3-13 deletion mutant

SupramoleculeName: Voltage-gated potassium channel Eag1 3-13 deletion mutant
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Supramolecule #3: calmodulin

SupramoleculeName: calmodulin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Potassium voltage-gated channel subfamily H member 1

MacromoleculeName: Potassium voltage-gated channel subfamily H member 1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 96.26143 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAQNTFLENI VRRSNDTNFV LGNAQIVDWP IVYSNDGFCK LSGYHRAEVM QKSSACSFMY GELTDKDTVE KVRQTFENYE MNSFEILMY KKNRTPVWFF VKIAPIRNEQ DKVVLFLCTF SDITAFKQPI EDDSCKGWGK FARLTRALTS SRGVLQQLAP S VQKGENVH ...String:
MAQNTFLENI VRRSNDTNFV LGNAQIVDWP IVYSNDGFCK LSGYHRAEVM QKSSACSFMY GELTDKDTVE KVRQTFENYE MNSFEILMY KKNRTPVWFF VKIAPIRNEQ DKVVLFLCTF SDITAFKQPI EDDSCKGWGK FARLTRALTS SRGVLQQLAP S VQKGENVH KHSRLAEVLQ LGSDILPQYK QEAPKTPPHI ILHYCVFKTT WDWIILILTF YTAILVPYNV SFKTRQNNVA WL VVDSIVD VIFLVDIVLN FHTTFVGPAG EVISDPKLIR MNYLKTWFVI DLLSCLPYDV INAFENVDEG ISSLFSSLKV VRL LRLGRV ARKLDHYIEY GAAVLVLLVC VFGLAAHWMA CIWYSIGDYE IFDEDTKTIR NNSWLYQLAL DIGTPYQFNG SGSG KWEGG PSKNSVYISS LYFTMTSLTS VGFGNIAPST DIEKIFAVAI MMIGSLLYAT IFGNVTTIFQ QMYANTNRYH EMLNS VRDF LKLYQVPKGL SERVMDYIVS TWSMSRGIDT EKVLQICPKD MRADICVHLN RKVFKEHPAF RLASDGCLRA LAMEFQ TVH CAPGDLIYHA GESVDSLCFV VSGSLEVIQD DEVVAILGKG DVFGDVFWKE ATLAQSCANV RALTYCDLHV IKRDALQ KV LEFYTAFSHS FSRNLILTYN LRKRIVFRKI SDVKREEEER MKRKNEAPLI LPPDHPVRRL FQRFRQQKEA RLAAERGG R DLDDLDVEKG NALTDHTSAN HSLVKASVVT VRESPATPVS FYPIPEQTLQ ATVLEVKHEL KEDIKALNAK MTSIEKQLS EILRILMSRG SSQSPQDTCE VSRPQSPESD RDIFGASSNS LEVLFQ

UniProtKB: Potassium voltage-gated channel subfamily H member 1, Potassium voltage-gated channel subfamily H member 1

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Macromolecule #2: Calmodulin-1

MacromoleculeName: Calmodulin-1 / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.852545 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREA FRVFDKDGNG YISAAELRHV MTNLGEKLTD EEVDEMIREA DIDGDGQVNY EEFVQMMTAK

UniProtKB: Calmodulin-1

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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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 54530
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5k7l


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

PDB-6pbx:
Single particle cryo-EM structure of the voltage-gated K+ channel Eag1 3-13 deletion mutant bound to calmodulin (conformation 2)

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