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- EMDB-5756: 3D EM Reconstruction of the AKAP18-PKA Complex in a Linear Confor... -

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Basic information

Entry
Database: EMDB / ID: EMD-5756
Title3D EM Reconstruction of the AKAP18-PKA Complex in a Linear Conformation
Map dataReconstruction of AKAP18-PKA in a Linear Conformation
Sample
  • Sample: AKAP18-PKA Complex in Linear Conformation
  • Protein or peptide: A-Kinase Anchoring Protein 18
  • Protein or peptide: A-Protein Kinase Catalytic Subunit
  • Protein or peptide: A-Protein Kinase Regulatory Subunit II alpha
KeywordsA-Kinase Anchoring Protein / AKAP / AKAP18 / A-Kinase / PKA / cAMP-Dependent Kinase / 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 / regulation of protein kinase A 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 / regulation of protein kinase A signaling / Hedgehog 'off' state / GPER1 signaling / 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 / nucleotide-activated protein kinase complex / RET signaling / positive regulation of potassium ion transmembrane transport / Ion homeostasis / Mitochondrial protein degradation / 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 / protein kinase A binding / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cellular response to cold / regulation of osteoblast differentiation / sperm capacitation / negative regulation of glycolytic process through fructose-6-phosphate / AMP binding / ciliary base / protein kinase A regulatory subunit binding / protein kinase A catalytic subunit binding / cAMP/PKA signal transduction / mesoderm formation / sperm flagellum / plasma membrane raft / axoneme / lateral plasma membrane / postsynaptic modulation of chemical synaptic transmission / cAMP binding / cellular response to cAMP / regulation of proteasomal protein catabolic process / negative regulation of TORC1 signaling / sperm midpiece / T-tubule / negative regulation of smoothened signaling pathway / positive regulation of gluconeogenesis / cellular response to glucagon stimulus / protein serine/threonine/tyrosine kinase activity / protein export from nucleus / acrosomal vesicle / hippocampal mossy fiber to CA3 synapse / sarcoplasmic reticulum / positive regulation of protein export from nucleus / 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 / manganese ion binding / cellular response to heat / peptidyl-serine phosphorylation / regulation of cell cycle / protein kinase activity / postsynapse / nuclear speck / apical plasma membrane / protein domain specific binding / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / ubiquitin protein ligase binding / synapse / protein kinase binding / perinuclear region of cytoplasm
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)
Methodsingle particle reconstruction / negative staining / Resolution: 35.0 Å
AuthorsSmith FD / Reichow SL / Esseltine JL / Shi D / Langeberg LK / Scott JD / 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, 2013-
Header (metadata) releaseOct 30, 2013-
Map releaseNov 13, 2013-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j4r
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5756.png

Downloads & links

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Map

FileDownload / File: emd_5756.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of AKAP18-PKA in a Linear Conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.2 Å/pix.
x 120 pix.
= 504. Å		4.2 Å/pix.
x 120 pix.
= 504. Å		4.2 Å/pix.
x 120 pix.
= 504. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-4.07958841 - 13.7339468
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 503.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z504.000504.000504.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-4.08013.734-0.000

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Supplemental data

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Sample components

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Entire : AKAP18-PKA Complex in Linear Conformation

EntireName: AKAP18-PKA Complex in Linear Conformation
Components
  • Sample: AKAP18-PKA Complex in Linear Conformation
  • Protein or peptide: A-Kinase Anchoring Protein 18
  • Protein or peptide: A-Protein Kinase Catalytic Subunit
  • Protein or peptide: A-Protein Kinase Regulatory Subunit II alpha

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Supramolecule #1000: AKAP18-PKA Complex in Linear Conformation

SupramoleculeName: AKAP18-PKA Complex in Linear Conformation / type: sample / ID: 1000
Oligomeric state: Hetero-pentamer composed of one AKAP18 bound to a dimer of the PKA Regulatory Subunit II and two copies of the PKA Catalytic Subunit
Number unique components: 3
Molecular weightExperimental: 240 KDa / Theoretical: 220 KDa / Method: Native PAGE

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Macromolecule #1: A-Kinase Anchoring Protein 18

MacromoleculeName: A-Kinase Anchoring Protein 18 / type: protein_or_peptide / ID: 1 / Name.synonym: AKAP18 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: Sf-9 / Recombinant plasmid: Bacmid

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Macromolecule #2: A-Protein Kinase Catalytic Subunit

MacromoleculeName: A-Protein Kinase Catalytic Subunit / type: protein_or_peptide / ID: 2 / Name.synonym: PKA C subunit / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)pLysS / Recombinant plasmid: pET15a

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Macromolecule #3: A-Protein Kinase Regulatory Subunit II alpha

MacromoleculeName: A-Protein Kinase Regulatory Subunit II alpha / type: protein_or_peptide / ID: 3 / Name.synonym: PKA RIIalpha Subunit / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)pLysS / Recombinant plasmid: pET28b

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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

Concentration0.01 mg/mL
BufferpH: 7.4
Details: 25 mM HEPES, 200 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol.
StainingType: NEGATIVE / Details: 0.75% (w/v) uranyl formate
GridDetails: 200 mesh copper grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureAverage: 298 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateMay 1, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Number real images: 1335 / Average electron dose: 15 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 50

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Image processing

DetailsImages were processed in IMAGIC and ISAC. 3D reconstruction was done in IMAGIC and FREALIGN.
CTF correctionDetails: Each Micrograph
Final reconstructionResolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, FREALIGN / Number images used: 1000

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Atomic model buiding 1

Initial modelPDB ID:

2izx


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsThis domain was linked to the AKAP18 central domain (PDB 2VFL) using COOT and they were fit together into the EM density.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j4r:
Pseudo-atomic model of the AKAP18-PKA Complex in a linear conformation derived from electron microscopy

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Atomic model buiding 2

Initial modelPDB ID:

2vfl


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThis domain was linked to the AKAP RII Binding Domain (PDB 2IZX) using COOT and they were fit together into the EM density.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j4r:
Pseudo-atomic model of the AKAP18-PKA Complex in a linear conformation derived from electron microscopy

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Atomic model buiding 3

Initial modelPDB ID:

2qvs


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsThis domain was linked to the PKA RIIalpha D/D domain (PDB 2IZX) using COOT and they were fit together into the EM density.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j4r:
Pseudo-atomic model of the AKAP18-PKA Complex in a linear conformation derived from electron microscopy

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