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- PDB-3j4r: Pseudo-atomic model of the AKAP18-PKA Complex in a linear conform... -

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Basic information

Entry
Database: PDB / ID: 3j4r
TitlePseudo-atomic model of the AKAP18-PKA Complex in a linear conformation derived from electron microscopy
Components
  • A-kinase anchor protein 18
  • cAMP-dependent protein kinase catalytic subunit alpha
  • cAMP-dependent protein kinase type II-alpha regulatory subunit
KeywordsTRANSFERASE / A-kinase anchoring protein / cAMP-Dependent Kinase / RII / PKA regulatory subunit II / phosphorylation / anchoring / intrinsic disorder
Function / homology
Function and homology information


cAMP-dependent protein kinase regulator activity / PKA activation in glucagon signalling / CREB1 phosphorylation through the activation of Adenylate Cyclase / HDL assembly / DARPP-32 events / Rap1 signalling / PKA activation / Regulation of insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GPER1 signaling ...cAMP-dependent protein kinase regulator activity / PKA activation in glucagon signalling / CREB1 phosphorylation through the activation of Adenylate Cyclase / HDL assembly / DARPP-32 events / Rap1 signalling / PKA activation / Regulation of insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GPER1 signaling / Hedgehog 'off' state / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / exocytic vesicle / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / MAPK6/MAPK4 signaling / GLI3 is processed to GLI3R by the proteasome / AURKA Activation by TPX2 / regulation of membrane repolarization / Factors involved in megakaryocyte development and platelet production / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Regulation of PLK1 Activity at G2/M Transition / Interleukin-3, Interleukin-5 and GM-CSF signaling / CD209 (DC-SIGN) signaling / RET signaling / nucleotide-activated protein kinase complex / positive regulation of potassium ion transmembrane transport / Mitochondrial protein degradation / Ion homeostasis / VEGFA-VEGFR2 Pathway / negative regulation of cAMP/PKA signal transduction / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / regulation of protein processing / cAMP-dependent protein kinase activity / protein localization to lipid droplet / cAMP-dependent protein kinase complex / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / protein kinase A binding / cellular response to cold / regulation of osteoblast differentiation / sperm capacitation / AMP binding / ciliary base / negative regulation of glycolytic process through fructose-6-phosphate / protein kinase A regulatory subunit binding / protein kinase A catalytic subunit binding / mesoderm formation / cAMP/PKA signal transduction / sperm flagellum / plasma membrane raft / axoneme / lateral plasma membrane / postsynaptic modulation of chemical synaptic transmission / cAMP binding / negative regulation of TORC1 signaling / cellular response to cAMP / regulation of proteasomal protein catabolic process / T-tubule / sperm midpiece / positive regulation of gluconeogenesis / protein serine/threonine/tyrosine kinase activity / cellular response to glucagon stimulus / protein export from nucleus / hippocampal mossy fiber to CA3 synapse / acrosomal vesicle / sarcoplasmic reticulum / positive regulation of protein export from nucleus / negative regulation of smoothened signaling pathway / neural tube closure / positive regulation of cholesterol biosynthetic process / modulation of chemical synaptic transmission / cellular response to glucose stimulus / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / neuromuscular junction / positive regulation of insulin secretion / adenylate cyclase-activating G protein-coupled receptor signaling pathway / mRNA processing / kinase activity / peptidyl-serine phosphorylation / manganese ion binding / cellular response to heat / postsynapse / protein kinase activity / regulation of cell cycle / nuclear speck / apical plasma membrane / protein domain specific binding / protein serine kinase activity / protein serine/threonine kinase activity / synapse / centrosome / ubiquitin protein ligase binding / protein kinase binding / perinuclear region of cytoplasm / glutamatergic synapse
Similarity search - Function
A-kinase anchor protein 7, RI-RII subunit-binding domain / PKA-RI-RII subunit binding domain of A-kinase anchor protein / : / Protein kinase A anchor protein, nuclear localisation signal domain / AKAP7 2'5' RNA ligase-like domain / cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / : ...A-kinase anchor protein 7, RI-RII subunit-binding domain / PKA-RI-RII subunit binding domain of A-kinase anchor protein / : / Protein kinase A anchor protein, nuclear localisation signal domain / AKAP7 2'5' RNA ligase-like domain / cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / : / Cyclic phosphodiesterase / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / cAMP-dependent protein kinase catalytic subunit / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / RmlC-like jelly roll fold / 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
cAMP-dependent protein kinase catalytic subunit alpha / cAMP-dependent protein kinase type II-alpha regulatory subunit / A-kinase anchor protein 7 isoforms delta and gamma / cAMP-dependent protein kinase type II-alpha regulatory subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 35 Å
AuthorsReichow, S.L. / Gonen, T.
CitationJournal: Elife / Year: 2013
Title: Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.
Authors: F Donelson Smith / Steve L Reichow / Jessica L Esseltine / Dan Shi / Lorene K Langeberg / John D Scott / Tamir Gonen /
Abstract: Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic ...Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.
History
DepositionSep 25, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2013Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 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: A-kinase anchor protein 18
B: cAMP-dependent protein kinase type II-alpha regulatory subunit
C: cAMP-dependent protein kinase type II-alpha regulatory subunit
D: cAMP-dependent protein kinase catalytic subunit alpha
E: cAMP-dependent protein kinase catalytic subunit alpha


Theoretical massNumber of molelcules
Total (without water)212,0245
Polymers212,0245
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein A-kinase anchor protein 18 / AKAP18


Mass: 39478.773 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): Sf-9 / References: UniProt: Q6JP77*PLUS
#2: Protein cAMP-dependent protein kinase type II-alpha regulatory subunit / Protein kinase / cAMP dependent regulatory / type II alpha / isoform CRA_b / cAMP-dependent protein ...Protein kinase / cAMP dependent regulatory / type II alpha / isoform CRA_b / cAMP-dependent protein kinase type II-alpha regulatory chain / cAMP-dependent protein kinase type II-alpha regulatory subunit


Mass: 45644.266 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Prkar2a, mCG_16488 / Plasmid: pET15a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: Q8K1M3, UniProt: P12367*PLUS
#3: Protein cAMP-dependent protein kinase catalytic subunit alpha / PKA C-alpha


Mass: 40628.555 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Prkaca, Pkaca / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: P05132, cAMP-dependent protein kinase
Sequence detailsAKAP18 IN THIS ENTRY IS FROM HOMO SAPIENS, BUT THE MODELED SEQUENCE IS FROM RATTUS NORVEGICUS (UNP Q6JP77).

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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

Component
IDNameTypeDetailsParent-ID
1AKAP18-PKA Complex in Linear ConformationCOMPLEXHetero-pentamer composed of one AKAP18 bound to a dimer of the PKA Regulatory Subunit II and two copies of the PKA Catalytic Subunit0
2A-Kinase Anchoring Protein 181
3A-Protein Kinase Regulatory Subunit II alpha1
4A-Protein Kinase Catalytic Subunit1
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.24 MDaYES
210.22 MDaNO
Buffer solutionName: 25 mM HEPES, pH 7.4, 200 mM NaCl, 0.5 mM EDTA, 1 mM DTT
pH: 7.4
Details: 25 mM HEPES, pH 7.4, 200 mM NaCl, 0.5 mM EDTA, 1 mM DTT
SpecimenConc.: 0.005 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO / Details: Stain 0.75% (w/v) uranyl formate
EM stainingType: NEGATIVE / Material: Uranyl Formate
Specimen supportDetails: 200 mesh copper grid with thin carbon support

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 12 / Date: May 1, 2012
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 52000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 6.3 mm
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: Single-Tilt / Temperature: 298 K / Tilt angle max: 50 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GENERIC GATAN
Image scansNum. digital images: 1335
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2FREALIGN3D reconstruction
3IMAGIC3D reconstruction
CTF correctionDetails: Each Micrograph
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Angular Reconstitution and Refinement / Resolution: 35 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 1000 / Nominal pixel size: 4.2 Å / Actual pixel size: 4.2 Å
Details: (Single particle details: Images were processed in IMAGIC and ISAC. 3D reconstruction was done in IMAGIC and FREALIGN.) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model building
IDProtocolSpaceTarget criteriaDetails
1RIGID BODY FITREALcross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--This domain was linked to the AKAP18 central domain (PDB 2VFL) using COOT and they were fit together into the EM density.
2RIGID BODY FITREALcross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--This domain was linked to the AKAP RII Binding Domain (PDB 2IZX) using COOT and they were fit together into the EM density.
3RIGID BODY FITREALcross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--This domain was linked to the PKA RIIalpha D/D domain (PDB 2IZX) using COOT and they were fit together into the EM density.
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-ID
12IZX

2izx

A12IZX1
22IZX

2izx

B12IZX1
32VFL

2vfl

A22VFL2
42QVS

2qvs

A32QVS3
52QVS

2qvs

B32QVS3
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms12903 0 0 0 12903

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