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- EMDB-41077: Cryo-EM structure of dodecameric CaMKII beta holoenzyme T287A T30... -

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Basic information

Entry
Database: EMDB / ID: EMD-41077
TitleCryo-EM structure of dodecameric CaMKII beta holoenzyme T287A T306A T307A
Map data
Sample
  • Complex: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A
    • Protein or peptide: Venus-tagged CaMKII beta holoenzyme mutant
KeywordsHigh-order oligomer / Protein Kinase / Signaling / Memory / SIGNALING PROTEIN
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 / regulation of neuron migration / Ca2+/calmodulin-dependent protein kinase / structural constituent of postsynaptic actin cytoskeleton / regulation of synapse maturation / positive regulation of dendritic spine morphogenesis / calcium-dependent protein serine/threonine kinase activity / Trafficking of AMPA receptors / RAF/MAP kinase cascade / calcium/calmodulin-dependent protein kinase activity / response to psychosocial stress / Ion homeostasis / 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 / generation of precursor metabolites and energy / long-term synaptic potentiation / apoptotic signaling pathway / 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 / peptidyl-serine phosphorylation / 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 / cytoplasm / cytosol
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 speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsChien C-T / Chiu W / Khan S
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41077.png

Downloads & links

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Map

FileDownload / File: emd_41077.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.68045616 - 1.0680436
Average (Standard dev.)-0.000670276 (±0.019983903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41077_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41077_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Venus-tagged CaMKII beta holoenzyme T287A T306A T307A

EntireName: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A
Components
  • Complex: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A
    • Protein or peptide: Venus-tagged CaMKII beta holoenzyme mutant

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Supramolecule #1: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A

SupramoleculeName: Venus-tagged CaMKII beta holoenzyme T287A T306A T307A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Venus-tagged CaMKII beta holoenzyme mutant

MacromoleculeName: Venus-tagged CaMKII beta holoenzyme mutant / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 90.846859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH VSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKLICTT GKLPVPWPTL VTTLGYGLQ CFARYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH K LEYNYNSH ...String:
MGSSHHHHHH SSGLVPRGSH VSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKLICTT GKLPVPWPTL VTTLGYGLQ CFARYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH K LEYNYNSH NVYITADKQK NGIKANFKIR HNIEDGGVQL ADHYQQNTPI GDGPVLLPDN HYLSYQSKLS KDPNEKRDHM VL LEFVTAA GITLGMDELY KSGLRSRAQA SNSAVDMATT VTCTRFTDEY QLYEDIGKGA FSVVRRCVKL CTGHEYAAKI INT KKLSAR DHQKLEREAR ICRLLKHSNI VRLHDSISEE GFHYLVFDLV TGGELFEDIV AREYYSEADA SHCIQQILEA VLHC HQMGV VHRDLKPENL LLASKCKGAA VKLADFGLAI EVQGDQQAWF GFAGTPGYLS PEVLRKEAYG KPVDIWACGV ILYIL LVGY PPFWDEDQHK LYQQIKAGAY DFPSPEWDTV TPEAKNLINQ MLTINPAKRI TAHEALKHPW VCQRSTVASM MHRQEA VEC LKKFNARRKL KGAILAAMLA TRNFSVGRQT TAPATMSTAA SGTTMGLVEQ AKSLLNKKAD GVKPQTNSTK NSSAITS PK GSLPPAALEP QTTVIHNPVD GIKESSDSTN TTIEDEDAKA RKQEIIKTTE QLIEAVNNGD FEAYAKICDP GLTSFEPE A LGNLVEGMDF HRFYFENLLA KNSKPIHTTI LNPHVHVIGE DAACIAYIRL TQYIDGQGRP RTSQSEETRV WHRPDGKWQ NVHFHCSGAP VAPLQ

UniProtKB: Green fluorescent protein, Calcium/calmodulin-dependent protein kinase type II subunit beta

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration20 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 332360
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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