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- EMDB-8215: Single particle cryo-EM structure of the voltage-gated K+ channel... -

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Entry
Database: EMDB / ID: EMD-8215
TitleSingle particle cryo-EM structure of the voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Map dataVoltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Sample
  • Organelle or cellular component: Voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
    • Organelle or cellular component: Voltage-gated potassium channel Eag1
      • Protein or peptide: Potassium voltage-gated channel subfamily H member 1
    • Organelle or cellular component: Calmodulin
      • Protein or peptide: Calmodulin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
KeywordsVoltage-gated potassium channel / calmodulin / Cryoelectron microscopy / Eag1 / METAL TRANSPORT-CALCIUM BINDING PROTEIN complex
Function / homology
Function and homology information


Voltage gated Potassium channels / potassium channel complex / : / : / positive regulation of cyclic-nucleotide phosphodiesterase activity / regulation of presynaptic cytosolic calcium ion concentration / : / establishment of protein localization to mitochondrial membrane / delayed rectifier potassium channel activity / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential ...Voltage gated Potassium channels / potassium channel complex / : / : / positive regulation of cyclic-nucleotide phosphodiesterase activity / regulation of presynaptic cytosolic calcium ion concentration / : / establishment of protein localization to mitochondrial membrane / delayed rectifier potassium channel activity / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / negative regulation of peptidyl-threonine phosphorylation / type 3 metabotropic glutamate receptor binding / nuclear inner membrane / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / positive regulation of DNA binding / PKA activation / CaMK IV-mediated phosphorylation of CREB / negative regulation of high voltage-gated calcium channel activity / response to corticosterone / positive regulation of peptidyl-threonine phosphorylation / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / phosphatidylinositol bisphosphate binding / Activation of RAC1 downstream of NMDARs / nitric-oxide synthase binding / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / regulation of synaptic vesicle exocytosis / presynaptic endocytosis / regulation of cardiac muscle cell action potential / parallel fiber to Purkinje cell synapse / positive regulation of ryanodine-sensitive calcium-release channel activity / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / startle response / negative regulation of ryanodine-sensitive calcium-release channel activity / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / calcineurin-mediated signaling / regulation of synaptic vesicle endocytosis / Ion transport by P-type ATPases / positive regulation of protein autophosphorylation / Uptake and function of anthrax toxins / Long-term potentiation / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / protein phosphatase activator activity / adenylate cyclase binding / regulation of ryanodine-sensitive calcium-release channel activity / DARPP-32 events / axolemma / Smooth Muscle Contraction / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / positive regulation of protein serine/threonine kinase activity / RHO GTPases activate IQGAPs / phosphatidylinositol 3-kinase binding / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / presynaptic cytosol / calcium channel inhibitor activity / cellular response to interferon-beta / Protein methylation / Activation of AMPK downstream of NMDARs / Ion homeostasis / activation of adenylate cyclase activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / enzyme regulator activity / regulation of calcium-mediated signaling / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / titin binding / voltage-gated potassium channel complex / 14-3-3 protein binding / potassium ion transmembrane transport / sperm midpiece / substantia nigra development / calcium channel complex / calyx of Held / nitric-oxide synthase regulator activity / FCERI mediated Ca+2 mobilization / response to amphetamine / positive regulation of nitric-oxide synthase activity / Ras activation upon Ca2+ influx through NMDA receptor / FCGR3A-mediated IL10 synthesis / adenylate cyclase activator activity / regulation of heart rate / cellular 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 ...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. / PAS domain / RmlC-like jelly roll fold / 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 / Calmodulin-3 / Voltage-gated delayed rectifier potassium channel KCNH1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 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: Science / Year: 2016
Title: Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.
Authors: Jonathan R Whicher / Roderick MacKinnon /
Abstract: Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the ...Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K(v) structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K(v) channels and provides a template to further understand the gating properties of Eag1 and related channels.
History
DepositionJun 16, 2016-
Header (metadata) releaseAug 17, 2016-
Map releaseAug 17, 2016-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.032
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.032
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k7l
  • Surface level: 0.032
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8215.png

Downloads & links

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Map

FileDownload / File: emd_8215.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVoltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.032 / Movie #1: 0.032
Minimum - Maximum-0.018570647 - 0.08381803
Average (Standard dev.)-0.0015307135 (±0.005304274)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0190.084-0.002

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Supplemental data

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Additional map: Voltage-gated K+ channel Eag1 bound to the channel...

Fileemd_8215_additional_1.map
AnnotationVoltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Voltage-gated K+ channel Eag1 bound to the channel...

Fileemd_8215_additional_2.map
AnnotationVoltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Voltage-gated K+ channel Eag1 bound to the channel...

Fileemd_8215_additional_3.map
AnnotationVoltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
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 K+ channel Eag1 bound to the channel inhibitor calm...

EntireName: Voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Components
  • Organelle or cellular component: Voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
    • Organelle or cellular component: Voltage-gated potassium channel Eag1
      • Protein or peptide: Potassium voltage-gated channel subfamily H member 1
    • Organelle or cellular component: Calmodulin
      • Protein or peptide: Calmodulin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Voltage-gated K+ channel Eag1 bound to the channel inhibitor calm...

SupramoleculeName: Voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 450 KDa

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Supramolecule #2: Voltage-gated potassium channel Eag1

SupramoleculeName: Voltage-gated potassium channel Eag1 / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat) / Location in cell: Cell membrane
Molecular weightTheoretical: 380 KDa

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Supramolecule #3: Calmodulin

SupramoleculeName: Calmodulin / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17 KDa

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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: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 97.359766 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTMAGGRRGL VAPQNTFLEN IVRRSNDTNF VLGNAQIVDW PIVYSNDGFC KLSGYHRAEV MQKSSACSFM YGELTDKDTV EKVRQTFEN YEMNSFEILM YKKNRTPVWF FVKIAPIRNE QDKVVLFLCT FSDITAFKQP IEDDSCKGWG KFARLTRALT S SRGVLQQL ...String:
MTMAGGRRGL VAPQNTFLEN IVRRSNDTNF VLGNAQIVDW PIVYSNDGFC KLSGYHRAEV MQKSSACSFM YGELTDKDTV EKVRQTFEN YEMNSFEILM YKKNRTPVWF FVKIAPIRNE QDKVVLFLCT FSDITAFKQP IEDDSCKGWG KFARLTRALT S SRGVLQQL APSVQKGENV HKHSRLAEVL QLGSDILPQY KQEAPKTPPH IILHYCVFKT TWDWIILILT FYTAILVPYN VS FKTRQNN VAWLVVDSIV DVIFLVDIVL NFHTTFVGPA GEVISDPKLI RMNYLKTWFV IDLLSCLPYD VINAFENVDE GIS SLFSSL KVVRLLRLGR VARKLDHYIE YGAAVLVLLV CVFGLAAHWM ACIWYSIGDY EIFDEDTKTI RNNSWLYQLA LDIG TPYQF NGSGSGKWEG GPSKNSVYIS SLYFTMTSLT SVGFGNIAPS TDIEKIFAVA IMMIGSLLYA TIFGNVTTIF QQMYA NTNR YHEMLNSVRD FLKLYQVPKG LSERVMDYIV STWSMSRGID TEKVLQICPK DMRADICVHL NRKVFKEHPA FRLASD GCL RALAMEFQTV HCAPGDLIYH AGESVDSLCF VVSGSLEVIQ DDEVVAILGK GDVFGDVFWK EATLAQSCAN VRALTYC DL HVIKRDALQK VLEFYTAFSH SFSRNLILTY NLRKRIVFRK ISDVKREEEE RMKRKNEAPL ILPPDHPVRR LFQRFRQQ K EARLAAERGG RDLDDLDVEK GNALTDHTSA NHSLVKASVV TVRESPATPV SFYPIPEQTL QATVLEVKHE LKEDIKALN AKMTSIEKQL SEILRILMSR GSSQSPQDTC EVSRPQSPES DRDIFGASSN SLEVLFQ

UniProtKB: Voltage-gated delayed rectifier potassium channel KCNH1, Voltage-gated delayed rectifier potassium channel KCNH1

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Macromolecule #2: Calmodulin

MacromoleculeName: Calmodulin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / 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-3

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
300.0 mMKClpotassium chloride
5.0 mMCaCl2calcium chloride
5.0 mMDTT
1.0 mMDodecyl Maltopyranoside
0.2 mMCholesteryl hemisuccinate
0.05 mg/mLPC:PE:PS (3:1:1)
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 86 % / Instrument: FEI VITROBOT MARK IV
Details: Plunged into liquid ethane (FEI VITROBOT MARK IV).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1657 / Average electron dose: 1.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
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: 240000
Details: Particles were auto-picked and manually inspected in RELION.
Startup modelType of model: INSILICO MODEL / In silico model: Initial model was generated in EMAN.
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN
Details: 2D classification, 3D classification, and refinement were performed in RELION. Final map generation was performed in FREALIGN.
Number images used: 145000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION (ver. 1.4), FREALIGN)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-5k7l:
Single particle cryo-EM structure of the voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin

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