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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
Function / homology
Function and homology information


Voltage gated Potassium channels / potassium channel complex / regulation of presynaptic cytosolic calcium ion concentration / delayed rectifier potassium channel activity / : / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / parallel fiber to Purkinje cell synapse / CaM pathway ...Voltage gated Potassium channels / potassium channel complex / regulation of presynaptic cytosolic calcium ion concentration / delayed rectifier potassium channel activity / : / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / parallel fiber to Purkinje cell synapse / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / nuclear inner membrane / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / regulation of synaptic vesicle endocytosis / phosphatidylinositol bisphosphate binding / 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 / regulation of synaptic vesicle exocytosis / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / response to corticosterone / positive regulation of ryanodine-sensitive calcium-release channel activity / Negative regulation of NMDA receptor-mediated neuronal transmission / nitric-oxide synthase binding / regulation of cell communication by electrical coupling involved in cardiac conduction / startle response / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / Long-term potentiation / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / adenylate cyclase binding / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / axolemma / detection of calcium ion / Smooth Muscle Contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / cellular response to interferon-beta / regulation of cardiac muscle contraction / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / positive regulation of DNA binding / Protein methylation / enzyme regulator activity / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / Activation of AMPK downstream of NMDARs / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Ion homeostasis / positive regulation of protein autophosphorylation / potassium ion transmembrane transport / sperm midpiece / response to amphetamine / calcium channel complex / activation of adenylate cyclase activity / substantia nigra development / cellular response to calcium ion / adenylate cyclase activator activity / regulation of membrane potential / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / monoatomic ion transmembrane transport / protein serine/threonine kinase activator activity / nitric-oxide synthase regulator activity / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / 14-3-3 protein binding / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers
Similarity search - Function
Potassium channel, voltage-dependent, EAG / Potassium channel, voltage-dependent, EAG/ELK/ERG / PAS-associated, C-terminal / PAC domain profile. / PAS domain / 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-associated, C-terminal / PAC domain profile. / PAS domain / 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. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Calmodulin-1 / Calmodulin-3 / 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: 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
Damon Runyon Cancer Research FoundationDRG-2212-15 United States
Howard Hughes Medical Institute (HHMI) 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-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing 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
Voxel sizeX=Y=Z: 1.3 Å
Density
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
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293 GnTI- / Recombinant plasmid: pEG BacMam

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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
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293 GnTI- / Recombinant plasmid: pEG BacMam

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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

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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

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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 / N-Acetylglucosamine

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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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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
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.
CTF correctionSoftware - Name: CTFFIND4 (ver. 4)
Startup modelType of model: INSILICO MODEL / In silico model: Initial model was generated in EMAN.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION (ver. 1.4), FREALIGN)
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
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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