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

Basic information

• Entry: Database: EMDB / ID: EMD-5756
• Title: 3D EM Reconstruction of the AKAP18-PKA Complex in a Linear Conformation
• Map data: Reconstruction 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

• Keywords: A-Kinase Anchoring Protein / AKAP / AKAP18 / A-Kinase / PKA / cAMP-Dependent Kinase / Kinase / RII / PKA Regulatory Subunit II / Phosphorylation / Anchoring / Intrinsic Disorder

Function / homology

Function:

spontaneous exocytosis of neurotransmitter / PKA activation in glucagon signalling / CREB1 phosphorylation through the activation of Adenylate Cyclase / negative regulation of meiotic cell cycle / HDL assembly / positive regulation of triglyceride catabolic process / DARPP-32 events / Rap1 signalling / PKA activation / Regulation of insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GPER1 signaling / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Hedgehog 'off' state / MAPK6/MAPK4 signaling / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / Recruitment of NuMA to mitotic centrosomes / exocytic vesicle / AURKA Activation by TPX2 / cAMP-dependent protein kinase regulator activity / GLI3 is processed to GLI3R by the proteasome / Factors involved in megakaryocyte development and platelet production / regulation of membrane repolarization / Interleukin-3, Interleukin-5 and GM-CSF signaling / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / CD209 (DC-SIGN) signaling / RET signaling / Regulation of PLK1 Activity at G2/M Transition / Mitochondrial protein degradation / VEGFA-VEGFR2 Pathway / nucleotide-activated protein kinase complex / positive regulation of potassium ion transmembrane transport / Ion homeostasis / regulation of cellular respiration / cAMP-dependent protein kinase inhibitor activity / beta-2 adrenergic receptor binding / histone H1-4S35 kinase activity / cAMP-dependent protein kinase / negative regulation of glycolytic process / regulation of protein processing / cAMP-dependent protein kinase activity / cellular response to parathyroid hormone stimulus / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cAMP-dependent protein kinase complex / interleukin-2-mediated signaling pathway / regulation of osteoblast differentiation / sperm capacitation / cellular response to cold / AMP binding / small molecule binding / ciliary base / protein kinase A regulatory subunit binding / protein kinase A binding / protein kinase A catalytic subunit binding / negative regulation of glycolytic process through fructose-6-phosphate / intracellular potassium ion homeostasis / TORC1 signaling / mesoderm formation / action potential / lateral plasma membrane / plasma membrane raft / axoneme / cAMP/PKA signal transduction / sperm flagellum / postsynaptic modulation of chemical synaptic transmission / cAMP binding / negative regulation of cAMP/PKA signal transduction / regulation of synaptic transmission, glutamatergic / regulation of proteasomal protein catabolic process / negative regulation of TORC1 signaling / protein serine/threonine/tyrosine kinase activity / lipid droplet / cellular response to glucagon stimulus / T-tubule / positive regulation of gluconeogenesis / positive regulation of phagocytosis / cellular response to nutrient levels / cellular response to cAMP / acrosomal vesicle / positive regulation of protein export from nucleus / hippocampal mossy fiber to CA3 synapse / sarcoplasmic reticulum / negative regulation of smoothened signaling pathway / neuromuscular junction / neural tube closure / cellular response to glucose stimulus / positive regulation of cholesterol biosynthetic process / modulation of chemical synaptic transmission / small GTPase binding / mRNA processing / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / kinase activity / manganese ion binding / cellular response to heat / adenylate cyclase-activating G protein-coupled receptor signaling pathway / presynapse

Domain/homology:

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

Component:

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

• Biological species: Homo sapiens (human) / Mus musculus (house mouse)
• Method: single particle reconstruction / negative staining / Resolution: 35.0 Å
• Authors: Smith FD / Reichow SL / Esseltine JL / Shi D / Langeberg LK / Scott JD / Gonen T
• Citation: Journal: 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 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

Deposition	Sep 25, 2013	-
Header (metadata) release	Oct 30, 2013	-
Map release	Nov 13, 2013	-
Update	Feb 17, 2016	-
Current status	Feb 17, 2016	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_5756.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstruction of AKAP18-PKA in a Linear Conformation
• Voxel size: X=Y=Z: 4.2 Å

Density

Contour Level	By AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum	-4.07958841 - 13.7339468
Average (Standard dev.)	0.0 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 120 120 120 Spacing 120 120 120
Cell	A=B=C: 503.99997 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	4.2	4.2	4.2
M x/y/z	120	120	120
origin x/y/z	0.000	0.000	0.000
length x/y/z	504.000	504.000	504.000
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	120	120	120
D min/max/mean	-4.080	13.734	-0.000

Supplemental data

Sample components

Entire : AKAP18-PKA Complex in Linear Conformation

• Entire: Name: 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

Supramolecule #1000: AKAP18-PKA Complex in Linear Conformation

• Supramolecule: Name: 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 weight: Experimental: 240 KDa / Theoretical: 220 KDa / Method: Native PAGE

Macromolecule #1: A-Kinase Anchoring Protein 18

• Macromolecule: Name: 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 weight: Theoretical: 40 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: Sf-9 / Recombinant plasmid: Bacmid

Macromolecule #2: A-Protein Kinase Catalytic Subunit

• Macromolecule: Name: 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 weight: Theoretical: 40 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)pLysS / Recombinant plasmid: pET15a

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

• Macromolecule: Name: 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 weight: Theoretical: 45 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)pLysS / Recombinant plasmid: pET28b

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.01 mg/mL
• Buffer: pH: 7.4 ; Details: 25 mM HEPES, 200 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol.
• Staining: Type: NEGATIVE / Details: 0.75% (w/v) uranyl formate
• Grid: Details: 200 mesh copper grid with thin carbon support
• Vitrification: Cryogen name: NONE / Instrument: OTHER

Electron microscopy

• Microscope: FEI TECNAI 12
• Temperature: Average: 298 K
• Alignment procedure: Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
• Date: May 1, 2012
• Image recording: Category: CCD / Film or detector model: GENERIC GATAN / Number real images: 1335 / Average electron dose: 15 e/Ų
• Tilt angle min: 0
• Electron beam: Acceleration voltage: 120 kV / Electron source: LAB6
• Electron optics: Illumination 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 stage: Specimen holder model: OTHER / Tilt angle max: 50

Image processing

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

Atomic model buiding 1

Initial model

PDB ID:

2izx

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B

• Software: Name: Chimera
• Details: This domain was linked to the AKAP18 central domain (PDB 2VFL) using COOT and they were fit together into the EM density.
• Refinement: Space: 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

Atomic model buiding 2

Initial model

PDB ID:

2vfl

Chain - Chain ID: A

• Software: Name: Chimera
• Details: This domain was linked to the AKAP RII Binding Domain (PDB 2IZX) using COOT and they were fit together into the EM density.
• Refinement: Space: 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

Atomic model buiding 3

Initial model

PDB ID:

2qvs

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B

• Software: Name: Chimera
• 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.
• Refinement: Space: 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