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- EMDB-72914: Local refinement of active human green opsin mutant E129Q in comp... -

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Entry
Database: EMDB / ID: EMD-72914
TitleLocal refinement of active human green opsin mutant E129Q in complex with chimeric G protein
Map dataLocal refinement of active human green opsin mutant E129Q in complex with chimeric G protein
Sample
  • Complex: Local refinement of active human green opsin mutant E129Q in complex with chimeric G protein
KeywordsG protein-coupled receptor / cone opsin / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsYao W / Fay JF / Farrens DL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R01 EY029343A United States
CitationJournal: Biophys J / Year: 2026
Title: Biophysical and structural analysis of human green cone opsin.
Authors: Weekie Yao / Jonathan F Fay / David L Farrens /
Abstract: We describe a straightforward method for purifying and optimizing human green cone opsin (GCO), which we then used for biophysical and structural studies of a GCO mutant, GCO. Our results show that ...We describe a straightforward method for purifying and optimizing human green cone opsin (GCO), which we then used for biophysical and structural studies of a GCO mutant, GCO. Our results show that in dark-state GCO, residue E129 enables long-wavelength light absorption, presumably by acting as the counterion for the protonated retinal Schiff base. Notably, the Schiff base pKa in dark-state GCO appears to be markedly lower (pKa ≈4) than in the rhodopsin equivalent, Rho (pKa ≈7), indicating distinct electrostatic environments at the retinal attachment site. Functional studies show that light-activated GCO decays more slowly and activates more G-protein than wild-type GCO (GCO). To identify the basis for these differences, we determined the structure of active GCO bound to a G-protein. We first developed a streamlined workflow to identify conditions that enhance GCO binding to G-proteins. This approach involved screening GCO binding to Gα-CT resin (beads bearing tethered Gα C-terminal peptides), followed by small-scale pull-down assays using 1D4 antibody beads to detect co-purification of GCO with a Venus-tagged mini-G-protein. Using the optimized conditions, we determined a 3.0-Å cryo-EM structure of the GCO-G-protein complex. Comparison with rhodopsin and our recent 3.0-Å structure of GCO reveals that the active-state architectures are largely similar, with several intriguing differences. Together, these results establish a generalizable, streamlined approach for biophysical and structural analysis of cone opsins and provide new mechanistic insight into the activation and signaling properties of GCO.
History
DepositionSep 28, 2025-
Header (metadata) releaseMay 20, 2026-
Map releaseMay 20, 2026-
UpdateMay 20, 2026-
Current statusMay 20, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72914.png

Downloads & links

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Map

FileDownload / File: emd_72914.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement of active human green opsin mutant E129Q in complex with chimeric G protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 288 pix.
= 264.384 Å		0.92 Å/pix.
x 288 pix.
= 264.384 Å		0.92 Å/pix.
x 288 pix.
= 264.384 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.918 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-7.7016244 - 12.254341
Average (Standard dev.)-0.0017814784 (±0.09962055)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 264.384 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_72914_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_72914_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Local refinement of active human green opsin mutant E129Q in comp...

EntireName: Local refinement of active human green opsin mutant E129Q in complex with chimeric G protein
Components
  • Complex: Local refinement of active human green opsin mutant E129Q in complex with chimeric G protein

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Supramolecule #1: Local refinement of active human green opsin mutant E129Q in comp...

SupramoleculeName: Local refinement of active human green opsin mutant E129Q in complex with chimeric G protein
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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: 7 / Component - Concentration: 20.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 37.2 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 198039
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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