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- EMDB-6258: Electron cryo-microscopy of the G protein effector, PDE6 -

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Basic information

Entry
Database: EMDB / ID: EMD-6258
TitleElectron cryo-microscopy of the G protein effector, PDE6
Map data3D reconstruction of the complex of the phosphodiesterase of rod photoreceptor cells (PDE6) with Ros-1 Fab (PDE6-Fab). C2 symmetry was imposed during the reconstruction.
Sample
  • Sample: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from the ROS-1 monoclonal antibody
  • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase
  • Protein or peptide: monoclonal antibody Fab fragment of ROS-1
Keywordsphosphodiesterase / photoreceptor.
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 / negative regulation of cAMP-mediated signaling / regulation of mitochondrion organization / response to stimulus / 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
GafA domain of cone phosphodiesterase 6C / 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 speciesBos taurus (cattle) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsZhang Z / He F / Constantine R / Baker ML / Baehr W / Schmid MF / Wensel TG / Agosto MA
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
DepositionFeb 2, 2015-
Header (metadata) releaseMar 18, 2015-
Map releaseApr 22, 2015-
UpdateSep 30, 2015-
Current statusSep 30, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jab
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbq
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6258.gif

Downloads & links

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Map

FileDownload / File: emd_6258.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the complex of the phosphodiesterase of rod photoreceptor cells (PDE6) with Ros-1 Fab (PDE6-Fab). C2 symmetry was imposed during the reconstruction.
Voxel sizeX=Y=Z: 1.81 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.75767982 - 1.83459306
Average (Standard dev.)0.01092006 (±0.10389578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 434.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.811.811.81
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z434.400434.400434.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.7581.8350.011

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Supplemental data

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Sample components

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Entire : Bovine rod PDE6 holoenzyme in complex with the Fab fragment from ...

EntireName: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from the ROS-1 monoclonal antibody
Components
  • Sample: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from the ROS-1 monoclonal antibody
  • Protein or peptide: Rod cGMP-specific 3',5'-cyclic phosphodiesterase
  • Protein or peptide: monoclonal antibody Fab fragment of ROS-1

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Supramolecule #1000: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from ...

SupramoleculeName: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from the ROS-1 monoclonal antibody
type: sample / ID: 1000 / Oligomeric state: Two Fab molecules bind to PDE6 / Number unique components: 2
Molecular weightTheoretical: 320 KDa

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Macromolecule #1: Rod cGMP-specific 3',5'-cyclic phosphodiesterase

MacromoleculeName: Rod cGMP-specific 3',5'-cyclic phosphodiesterase / type: protein_or_peptide / ID: 1 / Name.synonym: PDE6
Details: PDE6 holoenzyme contains PDE6a (UniProt P11541), PDE6b (UniProt P23439), and PDE6g (UniProt P04972).
Number of copies: 1 / Oligomeric state: dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: retina / Cell: photoreceptor / Organelle: outer segment / Location in cell: disk membrane
Molecular weightTheoretical: 200 KDa

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Macromolecule #2: monoclonal antibody Fab fragment of ROS-1

MacromoleculeName: monoclonal antibody Fab fragment of ROS-1 / type: protein_or_peptide / ID: 2
Details: Fab fragment was purified using immobilized protein L.
Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse / Tissue: Ascites fluid
Molecular weightTheoretical: 50 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5 / Details: 20 mM sodium phosphate, 150 mM sodium chloride
GridDetails: 400 mesh glow-discharged Quantifoil grids with 2.0 A holes
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK III
Method: Applied 3 uL sample per grid and blotted for 1 second before plunging.

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMax: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DetailsParallel beam illumination
DateAug 8, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: citfit (EMAN) for each particle
Final two d classificationNumber classes: 20
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: EMAN
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.
Number images used: 12373
DetailsImage processing was performed using EMAN. 21,100 particles were manually boxed from ice images and CTF-corrected using Ctfit. Initial models were generated from reference-free class averages and a cylindrical starting model. The two refined models were essentially similar. 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.

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Atomic model buiding 1

Initial modelPDB ID:

3ibj


Chain - Chain ID: B
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera and refined using "Fit in Map" within Chimera. The fitting of GFA(A,B) was confirmed using Folderhunter.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jab:
Domain organization and conformational plasticity of the G protein effector, PDE6

PDB-3jbq:
Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

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Atomic model buiding 2

Initial modelPDB ID:

3jwr


Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: D
SoftwareName: Chimera
DetailsThe domains were fitted by manual docking using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jab:
Domain organization and conformational plasticity of the G protein effector, PDE6

PDB-3jbq:
Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

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Atomic model buiding 3

Initial modelPDB ID:

3dba


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using Chimera and refined using "Fit in Map" within Chimera. The fitting of GFA(A,B) were confirmed using Folderhunter.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jab:
Domain organization and conformational plasticity of the G protein effector, PDE6

PDB-3jbq:
Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

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Atomic model buiding 4

Initial modelPDB ID:

12e8


Chain - #0 - Chain ID: H / Chain - #1 - Chain ID: L
SoftwareName: Chimera
DetailsThe domains were fitted by manual docking using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jab:
Domain organization and conformational plasticity of the G protein effector, PDE6

PDB-3jbq:
Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

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