+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6258 | |||||||||
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Title | Electron cryo-microscopy of the G protein effector, PDE6 | |||||||||
Map data | 3D 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 |
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Keywords | phosphodiesterase / 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 / Inactivation, recovery and regulation of the phototransduction cascade / negative regulation of cAMP-mediated signaling ...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 / Inactivation, recovery and regulation of the phototransduction cascade / negative regulation of cAMP-mediated signaling / positive regulation of G protein-coupled receptor signaling pathway / Activation of the phototransduction cascade / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of vascular permeability / cellular response to granulocyte macrophage colony-stimulating factor stimulus / 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 / 3',5'-cGMP-stimulated cyclic-nucleotide phosphodiesterase activity / photoreceptor outer segment membrane / 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 / regulation of cAMP-mediated signaling / cAMP-mediated signaling / visual perception / synaptic membrane / cellular response to mechanical stimulus / photoreceptor disc membrane / positive regulation of inflammatory response / presynaptic membrane / mitochondrial outer membrane / molecular adaptor activity / mitochondrial inner membrane / mitochondrial matrix / positive regulation of gene expression / perinuclear region of cytoplasm / Golgi apparatus / negative regulation of transcription by RNA polymerase II / signal transduction / endoplasmic reticulum / protein homodimerization activity / zinc ion binding / nucleus / metal ion binding / plasma membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Bos taurus (cattle) / Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.0 Å | |||||||||
Authors | Zhang Z / He F / Constantine R / Baker ML / Baehr W / Schmid MF / Wensel TG / Agosto MA | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6258.map.gz | 47.2 MB | EMDB map data format | |
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Header (meta data) | emd-6258-v30.xml emd-6258.xml | 15.7 KB 15.7 KB | Display Display | EMDB header |
Images | 400_6258.gif 80_6258.gif | 38 KB 3.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6258 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6258 | HTTPS FTP |
-Validation report
Summary document | emd_6258_validation.pdf.gz | 287.9 KB | Display | EMDB validaton report |
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Full document | emd_6258_full_validation.pdf.gz | 287.5 KB | Display | |
Data in XML | emd_6258_validation.xml.gz | 6.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6258 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6258 | HTTPS FTP |
-Related structure data
Related structure data | 3jabMC 3jbqMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6258.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D 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. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.81 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Bovine rod PDE6 holoenzyme in complex with the Fab fragment from ...
Entire | Name: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from the ROS-1 monoclonal antibody |
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Components |
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-Supramolecule #1000: Bovine rod PDE6 holoenzyme in complex with the Fab fragment from ...
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 320 KDa |
-Macromolecule #1: Rod cGMP-specific 3',5'-cyclic phosphodiesterase
Macromolecule | Name: 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 |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: bovine / Tissue: retina / Cell: photoreceptor / Organelle: outer segment / Location in cell: disk membrane |
Molecular weight | Theoretical: 200 KDa |
-Macromolecule #2: monoclonal antibody Fab fragment of ROS-1
Macromolecule | Name: 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 |
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Source (natural) | Organism: Mus musculus (house mouse) / synonym: mouse / Tissue: Ascites fluid |
Molecular weight | Theoretical: 50 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.5 / Details: 20 mM sodium phosphate, 150 mM sodium chloride |
Grid | Details: 400 mesh glow-discharged Quantifoil grids with 2.0 A holes |
Vitrification | Cryogen 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. |
-Electron microscopy
Microscope | JEOL 2010F |
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Temperature | Max: 94 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification. |
Specialist optics | Energy filter - Name: FEI |
Details | Parallel beam illumination |
Date | Aug 8, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 60000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | Image 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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CTF correction | Details: citfit (EMAN) for each particle |
Final reconstruction | Algorithm: 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 |
Final two d classification | Number classes: 20 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: B |
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Software | Name: Chimera |
Details | The 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-3jab: PDB-3jbq: |
-Atomic model buiding 2
Initial model | PDB ID: Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: D |
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Software | Name: Chimera |
Details | The domains were fitted by manual docking using Chimera. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-3jab: PDB-3jbq: |
-Atomic model buiding 3
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | The 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-3jab: PDB-3jbq: |
-Atomic model buiding 4
Initial model | PDB ID: Chain - #0 - Chain ID: H / Chain - #1 - Chain ID: L |
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Software | Name: Chimera |
Details | The domains were fitted by manual docking using Chimera. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-3jab: PDB-3jbq: |