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- EMDB-42237: Bovine rod phosphodiesterase 6 bound to two retinal transducin al... -

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Basic information

Entry
Database: EMDB / ID: EMD-42237
TitleBovine rod phosphodiesterase 6 bound to two retinal transducin alpha subunits
Map data
Sample
  • Complex: Bovine rod phosphodiesterase 6 bound to two retinal transducin alpha subunits
Keywordsphosphodiesterase / GPCR effector enzyme / SIGNALING PROTEIN / complex
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsAplin C / Cerione RA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)1R01EY034867-01 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6.
Authors: Cody Aplin / Richard A Cerione /
Abstract: Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein ...Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (Gα). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant Gα subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of Gα and the inhibitor vardenafil occupying the active sites on the PDEα and PDEβ subunits. We proposed Gα-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody Gα binds to PDE6 in a similar manner as observed when the antibody is present, does Gα activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 Gα-PDE6 complexes form with either recombinant or retinal Gα in the absence of the Gα antibody. We show that Gα binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables Gα to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting Gα-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of Gα for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.
History
DepositionOct 6, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42237.png

Downloads & links

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Map

FileDownload / File: emd_42237.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 256 pix.
= 343.04 Å		1.34 Å/pix.
x 256 pix.
= 343.04 Å		1.34 Å/pix.
x 256 pix.
= 343.04 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.46745068 - 1.6492679
Average (Standard dev.)-0.0017130944 (±0.05658512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 343.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42237_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_42237_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Bovine rod phosphodiesterase 6 bound to two retinal transducin al...

EntireName: Bovine rod phosphodiesterase 6 bound to two retinal transducin alpha subunits
Components
  • Complex: Bovine rod phosphodiesterase 6 bound to two retinal transducin alpha subunits

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Supramolecule #1: Bovine rod phosphodiesterase 6 bound to two retinal transducin al...

SupramoleculeName: Bovine rod phosphodiesterase 6 bound to two retinal transducin alpha subunits
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Bos taurus (cattle)

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 42479
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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