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- PDB-6pbx: Single particle cryo-EM structure of the voltage-gated K+ channel... -

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Basic information

Entry
Database: PDB / ID: 6pbx
TitleSingle particle cryo-EM structure of the voltage-gated K+ channel Eag1 3-13 deletion mutant bound to calmodulin (conformation 2)
Components
  • Calmodulin-1
  • Potassium voltage-gated channel subfamily H member 1
KeywordsTRANSPORT PROTEIN/CALCIUM BINDING PROTEIN / Voltage-gated potassium channel / ion channel / calmodulin / TRANSPORT PROTEIN-CALCIUM BINDING PROTEIN complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsWhicher, J.R. / MacKinnon, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM43949 United States
Howard Hughes Medical Institute 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, ...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 Report
SummaryFull reportAbout validation report
History
DepositionJun 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 18, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin-1
C: Potassium voltage-gated channel subfamily H member 1
E: Potassium voltage-gated channel subfamily H member 1
G: Potassium voltage-gated channel subfamily H member 1
D: Calmodulin-1
F: Calmodulin-1
H: Calmodulin-1


Theoretical massNumber of molelcules
Total (without water)452,4568
Polymers452,4568
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39740 Å2
ΔGint-334 kcal/mol
Surface area146940 Å2

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Components

#1: Protein/peptide
Potassium voltage-gated channel subfamily H member 1 / Ether-a-go-go potassium channel 1 / r-eag / Voltage-gated potassium channel subunit Kv10.1


Mass: 96261.430 Da / Num. of mol.: 4 / Fragment: UNP residues 14-773,888-962
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Kcnh1, Eag / Production host: Homo sapiens (human) / References: UniProt: Q63472
#2: Protein/peptide
Calmodulin-1 /


Mass: 16852.545 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM1, CALM, CAM, CAM1 / Production host: Homo sapiens (human) / References: UniProt: P0DP23

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulin1, 20MULTIPLE SOURCES
2Voltage-gated potassium channel Eag1 3-13 deletion mutant11RECOMBINANT
3calmodulin21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Homo sapiens (human)9606
Source (recombinant)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-ID
12
23
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54530 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5K7L

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