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- PDB-6y94: Ca2+-bound Calmodulin mutant N53I -

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Basic information

Entry
Database: PDB / ID: 6y94
TitleCa2+-bound Calmodulin mutant N53I
ComponentsCalmodulin
KeywordsMETAL BINDING PROTEIN / Mutant / metal-binding / signaling protein / CPVT
Function / homology
Function and homology information


CaM pathway / Cam-PDE 1 activation / 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 ...CaM pathway / Cam-PDE 1 activation / 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 / 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 / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / 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 / 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 / substantia nigra development / adenylate cyclase activator activity / 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 / 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 / spindle pole / response to calcium ion / RAS processing / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / Signaling by BRAF and RAF1 fusions / Inactivation, recovery and regulation of the phototransduction cascade / Platelet degranulation / myelin sheath / Ca2+ pathway / RAF/MAP kinase cascade / vesicle / transmembrane transporter binding / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway
Similarity search - Function
: / 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 / EF-hand domain pair
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsHolt, C. / Nielsen, L.H. / Lau, K. / Brohus, M. / Sorensen, A.B. / Larsen, K.T. / Sommer, C. / Petegem, F.V. / Overgaard, M.T. / Wimmer, R.
Funding support Denmark, European Union, 6items
OrganizationGrant numberCountry
Danish Council for Independent ResearchDFF-1323-00344 Denmark
Novo Nordisk FoundationNNF18OC0053032 Denmark
The Carlsberg Foundation Denmark
European Union (EU)261863European Union
The SparNord foundation Denmark
The Obel Family foundation Denmark
CitationJournal: J.Biol.Chem. / Year: 2020
Title: The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.
Authors: Holt, C. / Hamborg, L. / Lau, K. / Brohus, M. / Sorensen, A.B. / Larsen, K.T. / Sommer, C. / Van Petegem, F. / Overgaard, M.T. / Wimmer, R.
History
DepositionMar 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 10, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
Revision 1.4Jun 19, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,8815
Polymers16,7201
Non-polymers1604
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, Mass of mutant validated by mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area170 Å2
ΔGint-25 kcal/mol
Surface area10860 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 25structures with the lowest energy
RepresentativeModel #1target function

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Components

#1: Protein Calmodulin


Mass: 16720.404 Da / Num. of mol.: 1 / Mutation: N53I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM1, CALM, CAM, CAM1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta 2 / References: UniProt: P0DP23
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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
121isotropic12D 1H-13C HSQC aliphatic
131isotropic12D 1H-13C HSQC aromatic
141isotropic13D 1H-15N TOCSY
151isotropic13D CBCA(CO)NH
1131isotropic13D HN(CA)CB
1121isotropic13D HBHA(CO)NH
1111isotropic13D (H)CCH-TOCSY
1301isotropic13D (H)CCH-TOCSY
1101isotropic13D HNCA
191isotropic13D HNCO
181isotropic13D HN(CA)CO
171isotropic12D (HB)CB(CGCD)HD
161isotropic12D (HB)CB(CGCDCE)HE
1181isotropic23D 1H-15N NOESY
1171isotropic23D 1H-13C NOESY aliphatic
1161isotropic23D 1H-13C NOESY aromatic
2242isotropic115N-T1
2232isotropic115N-T2
2222isotropic1{1H}-15N NOE
2252isotropic1T2 relaxation dispersion
3263isotropic115N-T1
3273isotropic115N-T2
3283isotropic1{1H}-15N NOE
3293isotropic1T2 relaxation dispersion

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.56 mM [U-99% 13C; U-99% 15N] Calmodulin N53I, 2 mM HEPES, 100 mM potassium chloride, 10 mM calcium chloride, 50 mM sodium acetate, 2 mM sodium azide, 0.05 mM TSP, 95% H2O/5% D2OCaCaM-N53I-13C-15N95% H2O/5% D2O
solution21.0 mM [U-99% 15N] Calmodulin N53I, 2 mM HEPES, 10 mM potassium chloride, 10 mM calcium chloride, 2 mM sodium azide, 0.05 mM TSP, 95% H2O/5% D2OCaCaM-N53I-15N95% H2O/5% D2O
solution31.0 mM [U-99% 13C; U-99% 15N] Calmodulin, 2 mM HEPES, 10 mM potassium chloride, 10 mM calcium chloride, 2 mM sodium azide, 0.05 mM TSP, 95% H2O/5% D2OCaCaM-WT-13C-15N95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.56 mMCalmodulin N53I[U-99% 13C; U-99% 15N]1
2 mMHEPESnatural abundance1
100 mMpotassium chloridenatural abundance1
10 mMcalcium chloridenatural abundance1
50 mMsodium acetatenatural abundance1
2 mMsodium azidenatural abundance1
0.05 mMTSPnatural abundance1
1.0 mMCalmodulin N53I[U-99% 15N]2
2 mMHEPESnatural abundance2
10 mMpotassium chloridenatural abundance2
10 mMcalcium chloridenatural abundance2
2 mMsodium azidenatural abundance2
0.05 mMTSPnatural abundance2
1.0 mMCalmodulin[U-99% 13C; U-99% 15N]3
2 mMHEPESnatural abundance3
10 mMpotassium chloridenatural abundance3
10 mMcalcium chloridenatural abundance3
2 mMsodium azidenatural abundance3
0.05 mMTSPnatural abundance3
Sample conditions
Conditions-IDIonic strengthLabelpHPH errPressure (kPa)Temperature (K)
1182 mMconditions_16.570.051 atm298.1 K
242 mMconditions_26.570.051 atm298.1 K
342 mMconditions_36.570.051 atm298.1 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III6001
Bruker AVANCE III HDBrukerAVANCE III HD9002

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Processing

NMR software
NameVersionDeveloperClassification
YASARA14.12.2YASARA Biosciences GmbH Dr. Elmar Krieger Wagramer Strasse 25/3/45 1220 Vienna Austria / Europerefinement
CYANA3Guntert, Mumenthaler and Wuthrichstructure calculation
TALOSTALOS+Cornilescu, Delaglio and Baxstructure calculation
CARA1.8.4.2Keller and Wuthrichchemical shift assignment
CARA1.8.4.2Keller and Wuthrichpeak picking
TopSpin3.2Bruker Biospinprocessing
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 25 / Conformers submitted total number: 20

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