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- PDB-6w4p: CaMKII alpha-30 Cryo-EM reconstruction - Class B -

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Basic information

Entry
Database: PDB / ID: 6w4p
TitleCaMKII alpha-30 Cryo-EM reconstruction - Class B
Components(Calcium/calmodulin-dependent protein kinase type II subunit alpha) x 2
KeywordsSIGNALING PROTEIN/TRANSFERASE / Calcium calmodulin dependent protein kinase II / holoenzyme / splicing / SIGNALING PROTEIN / SIGNALING PROTEIN-TRANSFERASE complex
Function / homology
Function and homology information


peptidyl-threonine autophosphorylation / regulation of endocannabinoid signaling pathway / calcium- and calmodulin-dependent protein kinase complex / Ca2+/calmodulin-dependent protein kinase / negative regulation of hydrolase activity / dendritic spine development / regulation of neuron migration / regulation of neurotransmitter secretion / Trafficking of AMPA receptors / positive regulation of calcium ion transport ...peptidyl-threonine autophosphorylation / regulation of endocannabinoid signaling pathway / calcium- and calmodulin-dependent protein kinase complex / Ca2+/calmodulin-dependent protein kinase / negative regulation of hydrolase activity / dendritic spine development / regulation of neuron migration / regulation of neurotransmitter secretion / Trafficking of AMPA receptors / positive regulation of calcium ion transport / calcium/calmodulin-dependent protein kinase activity / Assembly and cell surface presentation of NMDA receptors / regulation of mitochondrial membrane permeability involved in apoptotic process / CaMK IV-mediated phosphorylation of CREB / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / Unblocking of NMDA receptors, glutamate binding and activation / Ion transport by P-type ATPases / regulation of neuronal synaptic plasticity / Long-term potentiation / Regulation of MECP2 expression and activity / HSF1-dependent transactivation / glutamate receptor binding / regulation of protein localization to plasma membrane / cellular response to interferon-beta / positive regulation of cardiac muscle cell apoptotic process / Ion homeostasis / Ras activation upon Ca2+ influx through NMDA receptor / response to ischemia / angiotensin-activated signaling pathway / positive regulation of receptor signaling pathway via JAK-STAT / RAF activation / cellular response to type II interferon / long-term synaptic potentiation / G1/S transition of mitotic cell cycle / Interferon gamma signaling / 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 ion transport / positive regulation of NF-kappaB transcription factor activity / kinase activity / Signaling by BRAF and RAF1 fusions / endocytic vesicle membrane / Ca2+ pathway / RAF/MAP kinase cascade / dendritic spine / calmodulin binding / neuron projection / postsynaptic density / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / protein homodimerization activity / mitochondrion / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Calcium/calmodulin-dependent protein kinase II, association-domain / Calcium/calmodulin dependent protein kinase II association domain / NTF2-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. ...Calcium/calmodulin-dependent protein kinase II, association-domain / Calcium/calmodulin dependent protein kinase II association domain / NTF2-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Calcium/calmodulin-dependent protein kinase type II subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsChao, L.H. / Stratton, M.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM123157 United States
CitationJournal: Sci Signal / Year: 2020
Title: Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.
Authors: Roman Sloutsky / Noelle Dziedzic / Matthew J Dunn / Rachel M Bates / Ana P Torres-Ocampo / Sivakumar Boopathy / Brendan Page / John G Weeks / Luke H Chao / Margaret M Stratton /
Abstract: Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes ...Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo-electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.
History
DepositionMar 11, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Calcium/calmodulin-dependent protein kinase type II subunit alpha
B: Calcium/calmodulin-dependent protein kinase type II subunit alpha
C: Calcium/calmodulin-dependent protein kinase type II subunit alpha
D: Calcium/calmodulin-dependent protein kinase type II subunit alpha
E: Calcium/calmodulin-dependent protein kinase type II subunit alpha
F: Calcium/calmodulin-dependent protein kinase type II subunit alpha
G: Calcium/calmodulin-dependent protein kinase type II subunit alpha
I: Calcium/calmodulin-dependent protein kinase type II subunit alpha
J: Calcium/calmodulin-dependent protein kinase type II subunit alpha
K: Calcium/calmodulin-dependent protein kinase type II subunit alpha
L: Calcium/calmodulin-dependent protein kinase type II subunit alpha
M: Calcium/calmodulin-dependent protein kinase type II subunit alpha
H: Calcium/calmodulin-dependent protein kinase type II subunit alpha


Theoretical massNumber of molelcules
Total (without water)697,24213
Polymers697,24213
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Calcium/calmodulin-dependent protein kinase type II subunit alpha / CaMK-II subunit alpha


Mass: 53633.953 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAMK2A, CAMKA, KIAA0968 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9UQM7, Ca2+/calmodulin-dependent protein kinase
#2: Protein Calcium/calmodulin-dependent protein kinase type II subunit alpha / CaMK-II subunit alpha


Mass: 53634.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CAMK2A, CAMKA, KIAA0968 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9UQM7, Ca2+/calmodulin-dependent protein kinase

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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

ComponentName: CaMKII alpha-30 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 s using PELCO easiGlow / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 96 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 52 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40329 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13SOA

3soa

13SOA1PDBexperimental model
25IG3

5ig3

15IG32PDBexperimental model

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