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- PDB-3jbq: Domain Organization and Conformational Plasticity of the G Protei... -

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Entry
Database: PDB / ID: 3jbq
TitleDomain Organization and Conformational Plasticity of the G Protein Effector, PDE6
Components
  • (IgG1-kappa 2E8 ...) x 2
  • (phosphodiesterase ...) x 2
  • GafA domain of cone phosphodiesterase 6C
  • GafB domain of phosphodiesterase 2A
KeywordsHYDROLASE/IMMUNE SYSTEM / Phosphodiesterase / photoreceptor / PDE6 / HYDROLASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


cGMP effects / Smooth Muscle Contraction / RHOBTB1 GTPase cycle / cyclic-nucleotide phosphodiesterase activity / GMP catabolic process / cellular response to macrophage colony-stimulating factor stimulus / 3',5'-cyclic-GMP phosphodiesterase / cellular response to cGMP / positive regulation of G protein-coupled receptor signaling pathway / Inactivation, recovery and regulation of the phototransduction cascade ...cGMP effects / Smooth Muscle Contraction / RHOBTB1 GTPase cycle / cyclic-nucleotide phosphodiesterase activity / GMP catabolic process / cellular response to macrophage colony-stimulating factor stimulus / 3',5'-cyclic-GMP phosphodiesterase / cellular response to cGMP / positive regulation of G protein-coupled receptor signaling pathway / Inactivation, recovery and regulation of the phototransduction cascade / Activation of the phototransduction cascade / positive regulation of vascular permeability / ion binding / cellular response to granulocyte macrophage colony-stimulating factor stimulus / negative regulation of vascular permeability / establishment of endothelial barrier / response to stimulus / negative regulation of cAMP-mediated signaling / regulation of mitochondrion organization / Ca2+ pathway / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / cGMP-stimulated cyclic-nucleotide phosphodiesterase activity / 3',5'-cyclic-nucleotide phosphodiesterase / negative regulation of cGMP-mediated signaling / cGMP-mediated signaling / cGMP catabolic process / 3',5'-cyclic-GMP phosphodiesterase activity / cAMP-mediated signaling / cGMP binding / 3',5'-cyclic-nucleotide phosphodiesterase activity / 3',5'-cyclic-AMP phosphodiesterase activity / regulation of cAMP-mediated signaling / visual perception / synaptic membrane / photoreceptor disc membrane / positive regulation of inflammatory response / cellular response to mechanical stimulus / presynaptic membrane / mitochondrial inner membrane / mitochondrial outer membrane / positive regulation of MAPK cascade / molecular adaptor activity / mitochondrial matrix / positive regulation of gene expression / perinuclear region of cytoplasm / Golgi apparatus / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / signal transduction / protein homodimerization activity / zinc ion binding / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / GAF domain / 3'5'-cyclic nucleotide phosphodiesterase / Domain present in phytochromes and cGMP-specific phosphodiesterases. / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain superfamily / 3'5'-cyclic nucleotide phosphodiesterase ...Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / GAF domain / 3'5'-cyclic nucleotide phosphodiesterase / Domain present in phytochromes and cGMP-specific phosphodiesterases. / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain / 3'5'-cyclic nucleotide phosphodiesterase, conserved site / 3'5'-cyclic nucleotide phosphodiesterase, catalytic domain superfamily / 3'5'-cyclic nucleotide phosphodiesterase / 3'5'-cyclic nucleotide phosphodiesterase domain signature. / 3'5'-cyclic nucleotide phosphodiesterase domain profile. / GAF domain / GAF-like domain superfamily / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma / cGMP-dependent 3',5'-cyclic phosphodiesterase / Cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha' / cGMP-specific 3',5'-cyclic phosphodiesterase
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 11 Å
AuthorsZhang, Z. / He, F. / Constantine, R. / Baker, M.L. / Baehr, W. / Schmid, M.F. / Wensel, T.G. / Agosto, M.A.
CitationJournal: J Biol Chem / Year: 2015
Title: Domain organization and conformational plasticity of the G protein effector, PDE6.
Authors: Zhixian Zhang / Feng He / Ryan Constantine / Matthew L Baker / Wolfgang Baehr / Michael F Schmid / Theodore G Wensel / Melina A Agosto /
Abstract: The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two ...The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration.
History
DepositionSep 17, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 30, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Deposited structure unit
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  • EMDB-6258
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
L: IgG1-kappa 2E8 light chain
H: IgG1-kappa 2E8 heavy chain
B: phosphodiesterase 5/6 chimera catalytic domain
D: phosphodiesterase 6 gamma subunit inhibitory peptide
C: GafB domain of phosphodiesterase 2A
F: phosphodiesterase 5/6 chimera catalytic domain
X: phosphodiesterase 6 gamma subunit inhibitory peptide
G: GafB domain of phosphodiesterase 2A
l: IgG1-kappa 2E8 light chain
h: IgG1-kappa 2E8 heavy chain
1: GafA domain of cone phosphodiesterase 6C
2: GafA domain of cone phosphodiesterase 6C


Theoretical massNumber of molelcules
Total (without water)260,03212
Polymers260,03212
Non-polymers00
Water0
1


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

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Components

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Phosphodiesterase ... , 2 types, 4 molecules BFDX

#3: Protein phosphodiesterase 5/6 chimera catalytic domain / cGMP-specific 3' / 5'-cyclic phosphodiesterase / cone cGMP-specific 3' / 5'-cyclic ...cGMP-specific 3' / 5'-cyclic phosphodiesterase / cone cGMP-specific 3' / 5'-cyclic phosphodiesterase subunit alpha' chimera


Mass: 38282.086 Da / Num. of mol.: 2 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: Q28156*PLUS
#4: Protein/peptide phosphodiesterase 6 gamma subunit inhibitory peptide / Retinal rod rhodopsin-sensitive cGMP 3' / 5'-cyclic phosphodiesterase subunit gamma / GMP-PDE gamma


Mass: 2185.436 Da / Num. of mol.: 2 / Fragment: UNP residues 70-87 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: P04972

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Protein , 2 types, 4 molecules CG12

#5: Protein GafB domain of phosphodiesterase 2A / cGMP-dependent 3' / 5'-cyclic phosphodiesterase


Mass: 20671.434 Da / Num. of mol.: 2 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: P14099*PLUS
#6: Protein GafA domain of cone phosphodiesterase 6C / cone cGMP-specific 3' / 5'-cyclic phosphodiesterase subunit alpha'


Mass: 21554.822 Da / Num. of mol.: 2 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: P52731*PLUS

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Antibody , 2 types, 4 molecules LlHh

#1: Antibody IgG1-kappa 2E8 light chain


Mass: 23540.990 Da / Num. of mol.: 2 / Fragment: Fab / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Tissue: Ascites fluid
#2: Antibody IgG1-kappa 2E8 heavy chain


Mass: 23781.471 Da / Num. of mol.: 2 / Fragment: Fab / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Tissue: Ascites fluid

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Details

Sequence detailsTHE IMAGED PDE6 WAS DERIVED FROM BOS TAURUS, BUT SOME MODELED SEQUENCES ARE FROM HOMO SAPIENS ...THE IMAGED PDE6 WAS DERIVED FROM BOS TAURUS, BUT SOME MODELED SEQUENCES ARE FROM HOMO SAPIENS (CHAINS B, C, F, G) OR GALLUS GALLUS (CHAINS 1, 2). THE PHOSPHODIESTERASE 2A STRUCTURE WAS DETERMINED WITH FULL-LENGTH PROTEIN, BUT ONLY RESIDUES 387-571 WERE USED IN THE FITTED MODEL (CHAINS C, G).

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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
1Bovine rod PDE6 holoenzyme in complex with the Fab fragment of monoclonal antibody ROS-1COMPLEXTwo Fab molecules bind to PDE60
2Rod cGMP-specific 3',5'-cyclic phosphodiesterase1
3Fab fragment of monoclonal antibody ROS-11
Molecular weightValue: 0.32 MDa / Experimental value: NO
Buffer solutionName: 20 mM sodium phosphate, 150 mM sodium chloride / pH: 7.5 / Details: 20 mM sodium phosphate, 150 mM sodium chloride
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh glow-discharged Quantifoil grids with 2.0 Angstrom holes
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 93 K / Humidity: 95 %
Details: Applied 3 uL of sample per grid and blotted for 1 second before plunging into liquid ethane (FEI VITROBOT MARK III).
Method: Applied 3 ul of sample per grid, blotted for 1 second before plunging

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Electron microscopy imaging

MicroscopyModel: JEOL 2010F / Date: Aug 8, 2011 / Details: Parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000 X / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 94 K
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
EM imaging opticsEnergyfilter name: FEI
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3EMAN3D reconstruction
CTF correctionDetails: by particle using CtFit
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: projection matching / Resolution: 11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12373 / Nominal pixel size: 1.81 Å / Actual pixel size: 1.81 Å
Details: A total of 21100 particles were picked from ice images and CTF-corrected using Ctfit. Initial three-dimensional models were generated from either reference-free class averages or from a ...Details: A total of 21100 particles were picked from ice images and CTF-corrected using Ctfit. Initial three-dimensional models were generated from either reference-free class averages or from a cylinder. The two models were essentially similar. Three noise-seeded models were generated and used as initial models in Multirefine. A model with two Ros-1 Fabs bound emerged from a population of 15000 particles and was subjected to further refinement using standard iterative projection matching, class averaging, and Fourier reconstruction. The final three-dimensional map with C2 symmetry was generated from 12373 particles.
Num. of class averages: 20 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: REFINEMENT PROTOCOL--rigid body refinement DETAILS--The domains were fit using Chimera. The model was refined using Phenix real-space refinement.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
13DBAB1
23IBJA1
33JWRB1
43JWRD1
51200000000H1
61200000000L1
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms17450 0 0 0 17450

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