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- EMDB-8514: 3D reconstruction of the CaMKIIa holoenzyme. -

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Basic information

Entry
Database: EMDB / ID: EMD-8514
Title3D reconstruction of the CaMKIIa holoenzyme.
Map dataCaMKII holoenzyme structure resolved by negative stain EM.Ca2+/calmodulin-dependent protein kinase II
Sample
  • Complex: Calcium-calmodulin dependent kinase II alpha
    • Protein or peptide: Calcium/calmodulin-dependent protein kinase type II subunit alpha
KeywordsCalcium/calmodulin-dependent kinase II (CaMKII) / cell signaling / calcium / calmodulin (CaM) / long-term potentiation (LTP) / long-term depression (LTD) / synaptic plasticity / cooperativity / electron microscopy (EM) / single particle reconstruction / intrinsic disorder / TRANSFERASE
Function / homology
Function and homology information


HSF1-dependent transactivation / regulation of synaptic vesicle docking / glutamatergic postsynaptic density / RAF activation / Ion transport by P-type ATPases / peptidyl-threonine autophosphorylation / regulation of endocannabinoid signaling pathway / calcium- and calmodulin-dependent protein kinase complex / calmodulin dependent kinase signaling pathway / Interferon gamma signaling ...HSF1-dependent transactivation / regulation of synaptic vesicle docking / glutamatergic postsynaptic density / RAF activation / Ion transport by P-type ATPases / peptidyl-threonine autophosphorylation / regulation of endocannabinoid signaling pathway / calcium- and calmodulin-dependent protein kinase complex / calmodulin dependent kinase signaling pathway / Interferon gamma signaling / calcium-dependent protein serine/threonine kinase activity / NMDA selective glutamate receptor signaling pathway / regulation of neuron migration / Ca2+/calmodulin-dependent protein kinase / regulation of neurotransmitter secretion / dendritic spine development / Trafficking of AMPA receptors / Ca2+ pathway / positive regulation of calcium ion transport / postsynaptic neurotransmitter receptor diffusion trapping / postsynaptic specialization membrane / presynaptic cytosol / negative regulation of hydrolase activity / calmodulin-dependent protein kinase activity / RAF/MAP kinase cascade / GTPase activating protein binding / dendrite morphogenesis / regulation of mitochondrial membrane permeability involved in apoptotic process / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / postsynaptic cytosol / regulation of neuronal synaptic plasticity / Ion homeostasis / positive regulation of cardiac muscle cell apoptotic process / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / cellular response to interferon-beta / regulation of protein localization to plasma membrane / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / response to ischemia / angiotensin-activated signaling pathway / G1/S transition of mitotic cell cycle / positive regulation of receptor signaling pathway via JAK-STAT / Schaffer collateral - CA1 synapse / cellular response to type II interferon / calcium ion transport / kinase activity / dendritic spine / postsynaptic density / calmodulin binding / neuron projection / axon / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / dendrite / neuronal cell body / glutamatergic synapse / synapse / protein homodimerization activity / mitochondrion / ATP binding / identical protein binding / metal ion 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 / 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 speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsMyers J / Reichow SL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS081248 United States
CitationJournal: Nat Commun / Year: 2017
Title: The CaMKII holoenzyme structure in activation-competent conformations.
Authors: Janette B Myers / Vincent Zaegel / Steven J Coultrap / Adam P Miller / K Ulrich Bayer / Steve L Reichow /
Abstract: The Ca/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, ...The Ca/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15-35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.
History
DepositionDec 9, 2016-
Header (metadata) releaseDec 28, 2016-
Map releaseJun 21, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5u6y
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8514.png

Downloads & links

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Map

FileDownload / File: emd_8514.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCaMKII holoenzyme structure resolved by negative stain EM.
Voxel sizeX=Y=Z: 4.37 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-1.1835607 - 0.77028763
Average (Standard dev.)0.0003138977 (±0.025755277)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 559.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.374.374.37
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z559.360559.360559.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-1.1840.7700.000

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Supplemental data

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Sample components

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Entire : Calcium-calmodulin dependent kinase II alpha

EntireName: Calcium-calmodulin dependent kinase II alpha
Components
  • Complex: Calcium-calmodulin dependent kinase II alpha
    • Protein or peptide: Calcium/calmodulin-dependent protein kinase type II subunit alpha

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Supramolecule #1: Calcium-calmodulin dependent kinase II alpha

SupramoleculeName: Calcium-calmodulin dependent kinase II alpha / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Full-length CaMKII alpha wild type
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 620 KDa

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

MacromoleculeName: Calcium/calmodulin-dependent protein kinase type II subunit alpha
type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: Ca2+/calmodulin-dependent protein kinase
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 52.281535 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MYQLFEELGK GAFSVVRRCV KVLAGQEYAA KIINTKKLSA RDHQKLEREA RICRLLKHPN IVRLHDSISE EGHHYLIFDL VTGGELFED IVAREYYSEA DASHCIQQIL EAVLHCHQMG VVHRDLKPEN LLLASKLKGA AVKLADFGLA IEVEGEQQAW F GFAGTPGY ...String:
MYQLFEELGK GAFSVVRRCV KVLAGQEYAA KIINTKKLSA RDHQKLEREA RICRLLKHPN IVRLHDSISE EGHHYLIFDL VTGGELFED IVAREYYSEA DASHCIQQIL EAVLHCHQMG VVHRDLKPEN LLLASKLKGA AVKLADFGLA IEVEGEQQAW F GFAGTPGY LSPEVLRKDP YGKPVDLWAC GVILYILLVG YPPFWDEDQH RLYQQIKAGA YDFPSPEWDT VTPEAKDLIN KM LTINPSK RITAAEALKH PWISHRSTVA SCMHRQETVD CLKKFNARRK LKGAILTTML ATRNFSGGKS GGNKKSDGVK ESS ESTNTT IEDEDTKVRK QEIIKVTEQL IEAISNGDFE SYTKMCDPGM TAFEPEALGN LVEGLDFHRF YFENLWSRNS KPVH TTILN PHIHLMGDES ACIAYIRITQ YLDAGGIPRT AQSEETRVWH RRDGKWQIVH FHRSGA

UniProtKB: Calcium/calmodulin-dependent protein kinase type II subunit alpha

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMHEPES
120.0 mMKCl
0.5 mMEGTA
StainingType: NEGATIVE / Material: Uranyl Formate / Details: 0.75% (wt/vol) uranyl formate
GridModel: Ted Pella / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
Details100 nM complex

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49000
Image recordingFilm or detector model: FEI EAGLE (2k x 2k) / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Particle selectionNumber selected: 16616
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

51g3

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: EMAN (ver. 2.12)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4)
Details: Only modeled the unstructured linker region, residues 300-345. The rest came from two other, previously published structures, namely 5IG3 and 2VZ6
Number images used: 2000

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Atomic model buiding 1

Initial model
PDB IDChain

51g3

residue_range: 345-472, source_name: PDB, initial_model_type: experimental model

2vz6

residue_range: 13-300, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT / Target criteria: Correlation Coefficient
Output model

PDB-5u6y:
Pseudo-atomic model of the CaMKIIa holoenzyme.

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