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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


voltage-gated ion channel activity involved in regulation of presynaptic membrane potential / regulation of presynaptic cytosolic calcium ion concentration / potassium channel complex / delayed rectifier potassium channel activity / phosphatidylinositol bisphosphate binding / parallel fiber to Purkinje cell synapse / nuclear inner membrane / startle response / axolemma / 14-3-3 protein binding ...voltage-gated ion channel activity involved in regulation of presynaptic membrane potential / regulation of presynaptic cytosolic calcium ion concentration / potassium channel complex / delayed rectifier potassium channel activity / phosphatidylinositol bisphosphate binding / parallel fiber to Purkinje cell synapse / nuclear inner membrane / startle response / axolemma / 14-3-3 protein binding / glycogen catabolic process / N-terminal myristoylation domain binding / regulation of cell communication by electrical coupling involved in cardiac conduction / inositol phosphate metabolic process / negative regulation of peptidyl-threonine phosphorylation / protein phosphatase activator activity / positive regulation of ryanodine-sensitive calcium-release channel activity / potassium ion transmembrane transport / regulation of rhodopsin mediated signaling pathway / detection of calcium ion / catalytic complex / voltage-gated potassium channel complex / adenylate cyclase binding / positive regulation of cyclic-nucleotide phosphodiesterase activity / adenylate cyclase activator activity / voltage-gated potassium channel activity / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / regulation of ion transmembrane transport / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / positive regulation of phosphoprotein phosphatase activity / Wnt signaling pathway, calcium modulating pathway / positive regulation of protein dephosphorylation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / go:0051186: / titin binding / substantia nigra development / calcium channel complex / sarcomere / regulation of ryanodine-sensitive calcium-release channel activity / regulation of heart rate / protein serine/threonine kinase activator activity / positive regulation of protein autophosphorylation / cellular response to calcium ion / enzyme regulator activity / postsynaptic density membrane / phosphatidylinositol-mediated signaling / ion transmembrane transport / regulation of synaptic vesicle exocytosis / spindle microtubule / regulation of membrane potential / positive regulation of peptidyl-threonine phosphorylation / integral component of presynaptic membrane / positive regulation of protein serine/threonine kinase activity / regulation of nitric-oxide synthase activity / regulation of cytokinesis / presynaptic membrane / calcium-mediated signaling / muscle contraction / response to calcium ion / microtubule cytoskeleton organization / spindle pole / platelet degranulation / Fc-epsilon receptor signaling pathway / early endosome membrane / disordered domain specific binding / regulation of cell population proliferation / ion channel binding / perikaryon / vesicle / calmodulin binding / MAPK cascade / centrosome / G protein-coupled receptor signaling pathway / synapse / axon / protein domain specific binding / neuronal cell body / dendrite / protein-containing complex binding / calcium ion binding / protein kinase binding / viral process / perinuclear region of cytoplasm / cell surface / integral component of plasma membrane / protein-containing complex / extracellular region / nucleoplasm / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm
EF-hand domain pair / Potassium voltage-gated channel subfamily H member 1 / PAS domain / Cyclic nucleotide-binding domain / PAS-associated, C-terminal / EF-hand domain / Potassium channel, voltage-dependent, EAG/ELK/ERG / Potassium channel, voltage-dependent, EAG / Ion transport domain / EF-hand domain pair ...EF-hand domain pair / Potassium voltage-gated channel subfamily H member 1 / PAS domain / Cyclic nucleotide-binding domain / PAS-associated, C-terminal / EF-hand domain / Potassium channel, voltage-dependent, EAG/ELK/ERG / Potassium channel, voltage-dependent, EAG / Ion transport domain / EF-hand domain pair / RmlC-like jelly roll fold / EF-Hand 1, calcium-binding site / Cyclic nucleotide-binding-like / Voltage-dependent channel domain superfamily / PAC motif / PAS domain superfamily / Calmodulin / Cyclic nucleotide-binding domain / Ion transport protein / 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 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.
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
Revision 1.2Nov 20, 2019Group: Author supporting evidence / 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
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
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
IDNameTypeEntity IDParent-IDSource
1Voltage-gated potassium channel Eag1 3-13 deletion mutant bound to calmodulinCOMPLEX#1-#20MULTIPLE SOURCES
2Voltage-gated potassium channel Eag1 3-13 deletion mutantCOMPLEX#11RECOMBINANT
3calmodulinCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
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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