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Basic information

Entry
Database: PDB / ID: 3jab
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 / 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 / calmodulin-activated dual specificity 3',5'-cyclic-GMP, 3',5'-cyclic-AMP phosphodiesterase activity / calmodulin-activated 3',5'-cyclic-GMP phosphodiesterase activity / cellular response to cGMP ...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 / calmodulin-activated dual specificity 3',5'-cyclic-GMP, 3',5'-cyclic-AMP phosphodiesterase activity / calmodulin-activated 3',5'-cyclic-GMP phosphodiesterase activity / cellular response to cGMP / Inactivation, recovery and regulation of the phototransduction cascade / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of G protein-coupled receptor signaling pathway / Activation of the phototransduction cascade / cellular response to granulocyte macrophage colony-stimulating factor stimulus / positive regulation of vascular permeability / negative regulation of vascular permeability / establishment of endothelial barrier / regulation of mitochondrion organization / cGMP-mediated signaling / Ca2+ pathway / positive regulation of epidermal growth factor receptor signaling pathway / photoreceptor outer segment membrane / 3',5'-cGMP-stimulated cyclic-nucleotide phosphodiesterase activity / 3',5'-cGMP-inhibited cyclic-nucleotide phosphodiesterase activity / 3',5'-cyclic-nucleotide phosphodiesterase / negative regulation of cGMP-mediated signaling / cGMP catabolic process / cGMP binding / 3',5'-cyclic-GMP phosphodiesterase activity / 3',5'-cyclic-AMP phosphodiesterase activity / cAMP-mediated signaling / visual perception / synaptic membrane / cellular response to mechanical stimulus / positive regulation of inflammatory response / photoreceptor disc membrane / presynaptic membrane / molecular adaptor activity / mitochondrial outer membrane / mitochondrial inner membrane / positive regulation of MAPK cascade / mitochondrial matrix / positive regulation of gene expression / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / Golgi apparatus / 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 / 3'5'-cyclic nucleotide phosphodiesterase / 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. ...Retinal cGMP phosphodiesterase, gamma subunit / Retinal cGMP phosphodiesterase, gamma subunit superfamily / Retinal cGMP phosphodiesterase, gamma subunit / 3'5'-cyclic nucleotide phosphodiesterase / 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 / Domain present in phytochromes and cGMP-specific phosphodiesterases. / GAF domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain / GAF-like domain superfamily
Similarity search - Domain/homology
3-ISOBUTYL-1-METHYLXANTHINE / GafA domain of cone phosphodiesterase 6C / Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma / cGMP-dependent 3',5'-cyclic phosphodiesterase / cGMP-specific 3',5'-cyclic phosphodiesterase
Similarity search - Component
Biological speciesBos taurus (domestic cattle)
Mus musculus (house mouse)
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
DepositionMay 26, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.2Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / struct_site
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 / _pdbx_entry_details.has_protein_modification / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-6258
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GafA domain of cone phosphodiesterase 6C
M: GafA domain of cone phosphodiesterase 6C
B: GafB domain of phosphodiesterase 2A
N: GafB domain of phosphodiesterase 2A
C: phosphodiesterase 5/6 chimera catalytic domain
D: phosphodiesterase 6 gamma subunit inhibitory peptide
O: phosphodiesterase 5/6 chimera catalytic domain
P: phosphodiesterase 6 gamma subunit inhibitory peptide
L: IgG1-kappa 2E8 light chain
H: IgG1-kappa 2E8 heavy chain
R: IgG1-kappa 2E8 light chain
Q: IgG1-kappa 2E8 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,45816
Polymers258,56912
Non-polymers8894
Water00
1


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

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Components

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Protein , 2 types, 4 molecules AMBN

#1: Protein GafA domain of cone phosphodiesterase 6C / cone cGMP-specific 3' / 5'-cyclic phosphodiesterase subunit alpha'


Mass: 20725.941 Da / Num. of mol.: 2 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: A0A0M3KKT0*PLUS
#2: 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 (domestic cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: P14099*PLUS

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Phosphodiesterase ... , 2 types, 4 molecules CODP

#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 (domestic 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 (domestic cattle) / Cell: photoreceptor / Cellular location: disk membrane / Organelle: outer segment / Tissue: retina / References: UniProt: P04972

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Antibody , 2 types, 4 molecules LRHQ

#5: 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
#6: Antibody IgG1-kappa 2E8 heavy chain


Mass: 23878.586 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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Non-polymers , 1 types, 4 molecules

#7: Chemical
ChemComp-IBM / 3-ISOBUTYL-1-METHYLXANTHINE


Mass: 222.244 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H14N4O2 / Comment: inhibitor, antagonist*YM

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Details

Has protein modificationY
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, N, O) OR GALLUS GALLUS (CHAINS A, M). THE PHOSPHODIESTERASE 2A STRUCTURE WAS DETERMINED WITH FULL-LENGTH PROTEIN, BUT ONLY RESIDUES 387-571 WERE USED IN THE FITTED MODEL (CHAINS B, N).

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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 from the ROS-1 monoclonal antibodyCOMPLEXTwo Fab molecules bind to PDE60
2Rod cGMP-specific 3',5'-cyclic phosphodiesterase1
3monoclonal antibody Fab fragment of 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 A holes
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 93 K / Humidity: 95 %
Details: Applied 3 uL sample per grid and blotted for 1 second before plunging into liquid ethane (FEI VITROBOT MARK III).
Method: Applied 3 uL sample per grid and 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 FIELD / Nominal magnification: 60000 X / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
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
1UCSF Chimeramodel fitting
2EMAN3D reconstruction
CTF correctionDetails: EMAN ctfit
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: projection match / 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 21,100 particles were picked from ice images and CTF corrected using Ctfit. After an initial 3D model was generated as described for PDE6, three noise-seeded models were generated ...Details: A total of 21,100 particles were picked from ice images and CTF corrected using Ctfit. After an initial 3D model was generated as described for PDE6, three noise-seeded models were generated and used as initial models in the Multirefine procedure. A model with two Ros-1 Fab bound with a population of ~15,000 particles emerged and was subjected to further refinement using standard iterative projection matching, class averaging, and Fourier reconstruction. The final 3D maps with C2 symmetry were generated from 12,373 particles.
Num. of class averages: 20 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALREFINEMENT PROTOCOL--rigid body DETAILS--Domains were separately fitted by manual docking and Fit in Map in Chimera. The fitting of GFA(A,B) was confirmed using Folderhunter.
2
3
4
5
6
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-ID
13DBA

3dba

B13DBA1
23IBJ

3ibj

A23IBJ2
33JWR

3jwr

B33JWR3
43JWR

3jwr

D43JWR3
51200000000H512000000004
61200000000L612000000004
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms17556 0 64 0 17620

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