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- PDB-5k7l: Single particle cryo-EM structure of the voltage-gated K+ channel... -

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Basic information

Entry
Database: PDB / ID: 5k7l
TitleSingle particle cryo-EM structure of the voltage-gated K+ channel Eag1 bound to the channel inhibitor calmodulin
Components
  • Calmodulin
  • Potassium voltage-gated channel subfamily H member 1
KeywordsMETAL TRANSPORT/CALCIUM BINDING PROTEIN / Voltage-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 / regulation of presynaptic cytosolic calcium ion concentration / : / establishment of protein localization to mitochondrial membrane / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / delayed rectifier potassium channel activity / type 3 metabotropic glutamate receptor binding / CaM pathway / Cam-PDE 1 activation ...Voltage gated Potassium channels / potassium channel complex / regulation of presynaptic cytosolic calcium ion concentration / : / establishment of protein localization to mitochondrial membrane / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / delayed rectifier potassium channel activity / type 3 metabotropic glutamate receptor binding / CaM pathway / Cam-PDE 1 activation / nuclear inner membrane / Sodium/Calcium exchangers / parallel fiber to Purkinje cell synapse / Calmodulin induced events / regulation of synaptic vesicle endocytosis / 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 / regulation of synaptic vesicle exocytosis / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / positive regulation of cyclic-nucleotide phosphodiesterase activity / organelle localization by membrane tethering / phosphatidylinositol bisphosphate binding / negative regulation of calcium ion export across plasma membrane / response to corticosterone / autophagosome membrane docking / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / positive regulation of DNA binding / positive regulation of ryanodine-sensitive calcium-release channel activity / nitric-oxide synthase binding / regulation of cell communication by electrical coupling involved in cardiac conduction / Synthesis of IP3 and IP4 in the cytosol / negative regulation of peptidyl-threonine phosphorylation / Negative regulation of NMDA receptor-mediated neuronal transmission / Phase 0 - rapid depolarisation / startle response / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / RHO GTPases activate PAKs / Ion transport by P-type ATPases / : / Uptake and function of anthrax toxins / Long-term potentiation / adenylate cyclase binding / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / axolemma / Smooth Muscle Contraction / regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / cellular response to interferon-beta / phosphatidylinositol 3-kinase binding / eNOS activation / Protein methylation / voltage-gated potassium channel complex / activation of adenylate cyclase activity / enzyme regulator activity / 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 / Ion homeostasis / : / titin binding / positive regulation of protein autophosphorylation / regulation of calcium-mediated signaling / 14-3-3 protein binding / sperm midpiece / calcium channel complex / potassium ion transmembrane transport / nitric-oxide synthase regulator activity / substantia nigra development / adenylate cyclase activator activity / response to amphetamine / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / sarcomere / cellular response to calcium ion / FCERI mediated Ca+2 mobilization / protein serine/threonine kinase activator activity / FCGR3A-mediated IL10 synthesis / regulation of membrane potential / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / positive regulation of nitric-oxide synthase activity / VEGFR2 mediated vascular permeability / regulation of cytokinesis
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 / : ...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
CHOLESTEROL HEMISUCCINATE / Calmodulin-1 / Calmodulin-3 / Voltage-gated delayed rectifier potassium channel KCNH1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 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: 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
DepositionMay 26, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 17, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2016Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Database references
Category: citation / em_image_scans ...citation / em_image_scans / em_software / pdbx_audit_support
Item: _citation.journal_id_CSD / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.5Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_oper_list
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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Assembly

Deposited unit
A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,9204
Polymers114,2122
Non-polymers7082
Water00
1
A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin
hetero molecules

A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin
hetero molecules

A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin
hetero molecules

A: Potassium voltage-gated channel subfamily H member 1
B: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)459,68116
Polymers456,8498
Non-polymers2,8328
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C4 (4 fold cyclic))

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Components

#1: Protein Potassium voltage-gated channel subfamily H member 1 / Voltage-gated potassium channel Eag1 / Ether-a-go-go potassium channel 1 / r-eag / Voltage-gated ...Voltage-gated potassium channel Eag1 / Ether-a-go-go potassium channel 1 / r-eag / Voltage-gated potassium channel subunit Kv10.1


Mass: 97359.766 Da / Num. of mol.: 1 / Fragment: UNP residues 1-773, 888-962
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Kcnh1, Eag / Plasmid: pEG BacMam / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q63472
#2: Protein Calmodulin / CaM


Mass: 16852.545 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: CALM1, CALM, CAM, CAM1, CALM2, CAM2, CAMB, CALM3, CALML2, CAM3, CAMC, CAMIII
Plasmid: pEG BacMam / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P62158, UniProt: P0DP23*PLUS
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
Has protein modificationY

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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 K+ channel Eag1 bound to the channel inhibitor calmodulinORGANELLE OR CELLULAR COMPONENT#1-#20MULTIPLE SOURCES
2Voltage-gated potassium channel Eag1ORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3CalmodulinORGANELLE OR CELLULAR COMPONENT#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.45 MDaNO
220.38 MDaNO
330.017 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
12Rattus norvegicus (Norway rat)10116Cell membrane
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
12Homo sapiens (human)9606HEK293 GnTI-pEG BacMam
23Homo sapiens (human)9606HEK293 GnTI-pEG BacMam
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2300 mMpotassium chlorideKCl1
35 mMcalcium chlorideCaCl21
45 mMDTT1
51 mMDodecyl Maltopyranoside1
60.2 mMCholesteryl hemisuccinate1
70.05 mg/mLPC:PE:PS (3:1:1)1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 86 % / Details: Plunged into liquid ethane (FEI VITROBOT MARK IV)

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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 FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1657

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Processing

SoftwareName: REFMAC / Version: 5.8.0088 / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
4CTFFIND44CTF correction
7Cootmodel fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11FREALIGNfinal Euler assignment
13FREALIGN3D reconstruction
14REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 240000
Details: Particles were auto-picked and manually inspected in RELION.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145000
Details: 2D classification, 3D classification, and refinement were performed in RELION. Final map generation was performed in FREALIGN.
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 3.78→165 Å / Cor.coef. Fo:Fc: 0.979 / SU B: 61.954 / SU ML: 0.725 / ESU R: 0.687
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.27167 --
obs0.27167 38517 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 301.016 Å2
Baniso -1Baniso -2Baniso -3
1--0.69 Å20 Å20 Å2
2---0.69 Å20 Å2
3---1.39 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0196322
ELECTRON MICROSCOPYr_bond_other_d0.0010.025630
ELECTRON MICROSCOPYr_angle_refined_deg1.1881.9468653
ELECTRON MICROSCOPYr_angle_other_deg0.909312766
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.7655839
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.6823.517236
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.40815841
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.0171524
ELECTRON MICROSCOPYr_chiral_restr0.0710.21024
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.027343
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021496
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it10.27233.1413368
ELECTRON MICROSCOPYr_mcbond_other10.26433.1413367
ELECTRON MICROSCOPYr_mcangle_it16.9549.7094203
ELECTRON MICROSCOPYr_mcangle_other16.94949.714204
ELECTRON MICROSCOPYr_scbond_it19.89532.2042954
ELECTRON MICROSCOPYr_scbond_other19.89132.2052955
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other26.61448.3494451
ELECTRON MICROSCOPYr_long_range_B_refined29.63911517
ELECTRON MICROSCOPYr_long_range_B_other29.63811518
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.78→3.878 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.689 2822 -
Rfree-0 -
obs--100 %

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