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- PDB-6but: Solution structure of full-length apo mammalian calmodulin bound ... -

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Basic information

Entry
Database: PDB / ID: 6but
TitleSolution structure of full-length apo mammalian calmodulin bound to the IQ motif of the human voltage-gated sodium channel NaV1.2
Components
  • Calmodulin-1
  • Sodium channel protein type 2 subunit alpha
KeywordsMEMBRANE PROTEIN / Calcium-Binding Protein / METAL TRANSPORT Ion channel Neuronal Molecular recognition
Function / homology
Function and homology information


intrinsic apoptotic signaling pathway in response to osmotic stress / nerve development / paranode region of axon / dentate gyrus development / node of Ranvier / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / CaM pathway / Cam-PDE 1 activation ...intrinsic apoptotic signaling pathway in response to osmotic stress / nerve development / paranode region of axon / dentate gyrus development / node of Ranvier / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / CaM pathway / Cam-PDE 1 activation / voltage-gated sodium channel activity / Sodium/Calcium exchangers / Calmodulin induced events / 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 / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / positive regulation of cyclic-nucleotide phosphodiesterase activity / organelle localization by membrane tethering / negative regulation of calcium ion export across plasma membrane / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / mitochondrion-endoplasmic reticulum membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / sodium ion transport / positive regulation of ryanodine-sensitive calcium-release channel activity / 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 / 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 / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / intercalated disc / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / neuronal action potential / Smooth Muscle Contraction / regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / sodium ion transmembrane transport / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / cellular response to interferon-beta / eNOS activation / Protein methylation / voltage-gated potassium channel complex / 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 / sperm midpiece / calcium channel complex / T-tubule / substantia nigra development / adenylate cyclase activator activity / myelination / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / protein serine/threonine kinase activator activity / VEGFR2 mediated vascular permeability / VEGFR2 mediated cell proliferation / regulation of cytokinesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / positive regulation of peptidyl-threonine phosphorylation / determination of adult lifespan / spindle microtubule / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of receptor signaling pathway via JAK-STAT / RAF activation / Transcriptional activation of mitochondrial biogenesis / positive regulation of protein serine/threonine kinase activity / Stimuli-sensing channels / cellular response to type II interferon / memory / Sensory perception of sweet, bitter, and umami (glutamate) taste / spindle pole
Similarity search - Function
Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / IQ motif profile. ...Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / IQ motif profile. / : / Voltage-dependent channel 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 / Sodium channel protein type 2 subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / molecular dynamics
AuthorsMahling, R. / Kilpatrick, A.M. / Shea, M.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM57001 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)T32 NS045549 United States
Citation
Journal: Structure / Year: 2021
Title: Na V 1.2 EFL domain allosterically enhances Ca 2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD.
Authors: Mahling, R. / Hovey, L. / Isbell, H.M. / Marx, D.C. / Miller, M.S. / Kilpatrick, A.M. / Weaver, L.D. / Yoder, J.B. / Kim, E.H. / Andresen, C.N.J. / Li, S. / Shea, M.A.
#1: Journal: Biomol NMR Assign / Year: 2017
Title: Backbone resonance assignments of complexes of human voltage-dependent sodium channel NaV1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.
Authors: Mahling, R. / Kilpatrick, A.M. / Shea, M.A.
History
DepositionDec 11, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Apr 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calmodulin-1
B: Sodium channel protein type 2 subunit alpha


Theoretical massNumber of molelcules
Total (without water)20,4042
Polymers20,4042
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 40structures with the lowest energy
RepresentativeModel #1lowest average pairwise backbone rmsd

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Components

#1: Protein Calmodulin-1


Mass: 16721.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM1, CALM, CAM, CAM1 / Plasmid: pT7-7 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0DP23
#2: Protein/peptide Sodium channel protein type 2 subunit alpha / HBSC II / Sodium channel protein brain II subunit alpha / Sodium channel protein type II subunit ...HBSC II / Sodium channel protein brain II subunit alpha / Sodium channel protein type II subunit alpha / Voltage-gated sodium channel subunit alpha Nav1.2


Mass: 3682.411 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: First four residues (GPGS) are part of a 3C protease cleavage site, and in the deposit have been numbered -4 to -1. NaV1.2 residue 1901 is residue 5 of the peptide.
Source: (gene. exp.) Homo sapiens (human) / Gene: SCN2A, NAC2, SCN2A1, SCN2A2 / Plasmid: pBG101 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q99250

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
121isotropic13D HN(CA)CB
131isotropic13D CBCA(CO)NH
141isotropic13D HNCO
151isotropic13D HACACO
161isotropic13D C(CO)NH
171isotropic13D H(CCO)NH
282isotropic22D 1H-13C HMQC
292isotropic23D (H)CCH-TOCSY
2102isotropic33D 1H-13C NOESY

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.950 mM U-99% C13, U-99% N15 Calmodulin, 0.950 mM U-99% C13, U-99% N15 voltage-gated sodium channel NaV1.2 IQ motif, 0.1 mM U-98% 2H EDTA, 100 mM KCl, 10 mM [U-99% 2H] imidazole, 0.01 % w/v sodium azide, 90% H2O/10% D2O13C_15N_CaM_NaV1290% H2O/10% D2O
solution20.95 mM U-99% C13, U-99% N15 Calmodulin, 0.95 mM U-99% C13, U-99% N15 voltage-gated sodium channel NaV1.2 IQ motif, 0.1 mM U-98% 2H EDTA, 100 mM potassium chloride, 10 mM [U-99% 2H] imidazole, 0.01 % w/v sodium azide, 100% D2O13C_15N_CaM_NaV12_2100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.950 mMCalmodulinU-99% C13, U-99% N151
0.950 mMvoltage-gated sodium channel NaV1.2 IQ motifU-99% C13, U-99% N151
0.1 mMEDTAU-98% 2H1
100 mMKClnatural abundance1
10 mMimidazole[U-99% 2H]1
0.01 % w/vsodium azidenatural abundance1
0.95 mMCalmodulinU-99% C13, U-99% N152
0.95 mMvoltage-gated sodium channel NaV1.2 IQ motifU-99% C13, U-99% N152
0.1 mMEDTAU-98% 2H2
100 mMpotassium chloridenatural abundance2
10 mMimidazole[U-99% 2H]2
0.01 % w/vsodium azidenatural abundance2
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
1100 mMConditions_16.51 atm298 K
2100 mMConditions_26.51 atm298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Varian INOVAVarianINOVA6001
Bruker AVANCE IIBrukerAVANCE II5002
Bruker AVANCE IIBrukerAVANCE II8003

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Processing

NMR software
NameVersionDeveloperClassification
VNMRVariancollection
TopSpinBruker Biospincollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Analysis2.4CCPNpeak picking
Analysis2.4CCPNchemical shift assignment
CYANA2.1Guntert, Mumenthaler and Wuthrichstructure calculation
AmberAMBERTOOLS 17Case, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
RefinementMethod: molecular dynamics / Software ordinal: 8
NMR representativeSelection criteria: lowest average pairwise backbone rmsd
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 40 / Conformers submitted total number: 20

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