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- PDB-8t6q: Cryo-EM structure of dodecameric CaMKII beta holoenzyme T287A T30... -

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Basic information

Entry
Database: PDB / ID: 8t6q
TitleCryo-EM structure of dodecameric CaMKII beta holoenzyme T287A T306A T307A
ComponentsVenus-tagged CaMKII beta holoenzyme mutant
KeywordsSIGNALING PROTEIN / High-order oligomer / Protein Kinase / Signaling / Memory
Function / homology
Function and homology information


regulation of synaptic transmission, cholinergic / activation of meiosis involved in egg activation / cell projection morphogenesis / HSF1-dependent transactivation / RAF activation / Ion transport by P-type ATPases / hippocampal neuron apoptotic process / calcium- and calmodulin-dependent protein kinase complex / cellular response to homocysteine / Interferon gamma signaling ...regulation of synaptic transmission, cholinergic / activation of meiosis involved in egg activation / cell projection morphogenesis / HSF1-dependent transactivation / RAF activation / Ion transport by P-type ATPases / hippocampal neuron apoptotic process / calcium- and calmodulin-dependent protein kinase complex / cellular response to homocysteine / Interferon gamma signaling / positive regulation of synapse maturation / calcium-dependent protein serine/threonine kinase activity / Ca2+/calmodulin-dependent protein kinase / structural constituent of postsynaptic actin cytoskeleton / regulation of neuron migration / positive regulation of dendritic spine morphogenesis / Trafficking of AMPA receptors / regulation of synapse maturation / calcium/calmodulin-dependent protein kinase activity / RAF/MAP kinase cascade / Ion homeostasis / response to psychosocial stress / phospholipase binding / neuromuscular process controlling balance / Unblocking of NMDA receptors, glutamate binding and activation / regulation of protein localization to plasma membrane / spindle midzone / response to cadmium ion / sarcoplasmic reticulum membrane / bioluminescence / apoptotic signaling pathway / generation of precursor metabolites and energy / long-term synaptic potentiation / positive regulation of apoptotic signaling pathway / regulation of long-term neuronal synaptic plasticity / positive regulation of neuron projection development / G1/S transition of mitotic cell cycle / calcium ion transport / nervous system development / perikaryon / protein autophosphorylation / postsynaptic density / cell differentiation / calmodulin binding / neuron projection / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / glutamatergic synapse / dendrite / protein kinase binding / protein homodimerization activity / ATP binding / identical protein binding / 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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain ...Calcium/calmodulin-dependent protein kinase II, association-domain / Calcium/calmodulin dependent protein kinase II association domain / NTF2-like domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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 beta / Green fluorescent protein
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsChien, C.-T. / Chiu, W. / Khan, S.
Funding support United States, 1items
OrganizationGrant numberCountry
Other governmentU24 GM129541 United States
CitationJournal: Commun Biol / Year: 2024
Title: Hub stability in the calcium calmodulin-dependent protein kinase II.
Authors: Chih-Ta Chien / Henry Puhl / Steven S Vogel / Justin E Molloy / Wah Chiu / Shahid Khan /
Abstract: The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within ...The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.
History
DepositionJun 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Venus-tagged CaMKII beta holoenzyme mutant
B: Venus-tagged CaMKII beta holoenzyme mutant
C: Venus-tagged CaMKII beta holoenzyme mutant
D: Venus-tagged CaMKII beta holoenzyme mutant
F: Venus-tagged CaMKII beta holoenzyme mutant
G: Venus-tagged CaMKII beta holoenzyme mutant
H: Venus-tagged CaMKII beta holoenzyme mutant
I: Venus-tagged CaMKII beta holoenzyme mutant
J: Venus-tagged CaMKII beta holoenzyme mutant
K: Venus-tagged CaMKII beta holoenzyme mutant
L: Venus-tagged CaMKII beta holoenzyme mutant
N: Venus-tagged CaMKII beta holoenzyme mutant


Theoretical massNumber of molelcules
Total (without water)1,090,16212
Polymers1,090,16212
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Venus-tagged CaMKII beta holoenzyme mutant


Mass: 90846.859 Da / Num. of mol.: 12 / Mutation: T287A,T306A,T307A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Rattus norvegicus (Norway rat)
Gene: GFP, Camk2b / Production host: Escherichia coli (E. coli) / References: UniProt: P42212, UniProt: P08413

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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: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 1500 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 332360 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 88.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003113284
ELECTRON MICROSCOPYf_angle_d0.540218036
ELECTRON MICROSCOPYf_chiral_restr0.04191920
ELECTRON MICROSCOPYf_plane_restr0.00522400
ELECTRON MICROSCOPYf_dihedral_angle_d5.1211752

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