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- PDB-9ygz: Cryo-EM structure of active mutant human green cone opsin (E129Q)... -

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Entry
Database: PDB / ID: 9ygz
TitleCryo-EM structure of active mutant human green cone opsin (E129Q) in complex with chimeric G protein (miniGist)
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Genome polyprotein,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
  • Medium-wave-sensitive opsin 1
  • scFv16
KeywordsSIGNALING PROTEIN / G protein-coupled receptor / cone opsin / MEMBRANE PROTEIN
Function / homology
Function and homology information


Defective visual phototransduction due to OPN1MW loss of function / The retinoid cycle in cones (daylight vision) / Opsins / absorption of visible light / G protein-coupled photoreceptor activity / cellular response to light stimulus / sensory perception of chemical stimulus / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / beta-2 adrenergic receptor binding ...Defective visual phototransduction due to OPN1MW loss of function / The retinoid cycle in cones (daylight vision) / Opsins / absorption of visible light / G protein-coupled photoreceptor activity / cellular response to light stimulus / sensory perception of chemical stimulus / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / beta-2 adrenergic receptor binding / host cell mitochondrial membrane / host cell lipid droplet / symbiont-mediated transformation of host cell / symbiont-mediated suppression of host TRAF-mediated signal transduction / positive regulation of cytokinesis / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / photoreceptor activity / PKA activation in glucagon signalling / phototransduction / developmental growth / photoreceptor outer segment / D1 dopamine receptor binding / Hedgehog 'off' state / insulin-like growth factor receptor binding / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / visual perception / ionotropic glutamate receptor binding / bioluminescence / adenylate cyclase activator activity / generation of precursor metabolites and energy / bone development / platelet aggregation / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G beta:gamma signalling through BTK / photoreceptor disc membrane / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / ribonucleoside triphosphate phosphatase activity / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / heterotrimeric G-protein complex / positive regulation of cold-induced thermogenesis / G alpha (12/13) signalling events / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / extracellular vesicle / sensory perception of taste / Thrombin signalling through proteinase activated receptors (PARs) / signaling receptor complex adaptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / viral nucleocapsid / channel activity / retina development in camera-type eye / GTPase binding / G protein activity / fibroblast proliferation / Ca2+ pathway / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / clathrin-dependent endocytosis of virus by host cell / monoatomic ion transmembrane transport / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / RNA helicase activity / apical plasma membrane / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / G protein-coupled receptor signaling pathway / ribonucleoprotein complex
Similarity search - Function
Opsin red/green sensitive / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 ...Opsin red/green sensitive / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / Hepatitus C virus, Non-structural 5a protein, C-terminal / Hepatitis C virus NS5A, 1B domain superfamily / Hepatitis C virus non-structural protein NS2, C-terminal domain / Hepatitis C virus non-structural protein NS2, N-terminal domain / Hepatitis C virus non-structural protein NS2 / HCV NS5a protein C-terminal region / Hepatitis C virus, Non-structural protein NS4b / Hepatitis C virus, Core protein, N-terminal / Hepatitis C virus core protein, chain A superfamily / : / Hepatitis C virus non-structural protein NS4b / Hepatitis C virus capsid protein / Hepatitis C virus, Non-structural protein NS2 / Hepatitis C virus, Non-structural 5a protein / Hepatitis C virus, Non-structural 5a protein, domain 1a / Hepatitis C virus non-structural 5a, 1B domain / NS5A domain 1a superfamily / : / Hepatitis C virus non-structural 5a protein membrane anchor / Hepatitis C virus non-structural 5a zinc finger domain / Hepatitis C virus non-structural 5a domain 1b / NS3 RNA helicase, C-terminal helical domain / Hepacivirus nonstructural protein 2 (NS2) protease domain profile. / Hepatitis C virus, Non-structural protein NS4a / Hepatitis C virus non-structural protein NS4a / Hepatitis C virus, Core protein, C-terminal / Hepatitis C virus core protein / Hepatitis C virus, Non-structural protein E2/NS1 / Hepatitis C virus non-structural protein E2/NS1 / Hepatitis C virus, Envelope glycoprotein E1 / Hepatitis C virus envelope glycoprotein E1 / RNA dependent RNA polymerase, hepatitis C virus / Viral RNA dependent RNA polymerase / Hepatitis C virus, NS3 protease, Peptidase S29 / Hepatitis C virus NS3 protease / Hepacivirus/Pegivirus NS3 protease domain profile. / G-protein alpha subunit, group S / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / DEAD box, Flavivirus / Flavivirus DEAD domain / Serpentine type 7TM GPCR chemoreceptor Srsx / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Reverse transcriptase/Diguanylate cyclase domain / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / WD40/YVTN repeat-like-containing domain superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RETINAL / Medium-wave-sensitive opsin 1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas / Genome polyprotein
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsYao, W. / Fay, J.F. / Farrens, D.L.
Funding support United States, 1items
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 29, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2026Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 20, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Medium-wave-sensitive opsin 1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
H: scFv16
A: Genome polyprotein,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas
hetero molecules


Theoretical massNumber of molelcules
Total (without water)173,8646
Polymers173,5805
Non-polymers2841
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules RA

#1: Protein Medium-wave-sensitive opsin 1 / Green cone photoreceptor pigment / Green-sensitive opsin / GOP


Mass: 40784.414 Da / Num. of mol.: 1 / Mutation: E129Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OPN1MW, GCP / Plasmid: pMT4 / Cell (production host): fibroblast / Cell line (production host): COS-1 / Production host: Chlorocebus aethiops (grivet monkey) / Tissue (production host): kidney / References: UniProt: P04001
#5: Protein Genome polyprotein,Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas / Adenylate cyclase-stimulating G alpha protein / Extra large alphas protein / XLalphas


Mass: 57380.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pcDNA3 / Gene: GNAS, GNAS1 / Cell (production host): fibroblast / Cell line (production host): COS-1 / Production host: Chlorocebus aethiops (grivet monkey) / Tissue (production host): kidney
References: UniProt: W8GG88, UniProt: Q5JWF2, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BG

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38878.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Plasmid: pcDNA3 / Cell (production host): fibroblast / Cell line (production host): COS-1 / Production host: Chlorocebus aethiops (grivet monkey) / Tissue (production host): kidney / References: UniProt: P62873
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Plasmid: pcDNA3 / Cell (production host): fibroblast / Cell line (production host): COS-1 / Production host: Chlorocebus aethiops (grivet monkey) / Tissue (production host): kidney / References: UniProt: P59768

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Antibody / Non-polymers , 2 types, 2 molecules H

#4: Antibody scFv16


Mass: 28674.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
#6: Chemical ChemComp-RET / RETINAL


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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

ComponentName: Cryo-EM structure of active mutant human green cone opsin (E129Q) in complex with chimeric G protein (miniGist)
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Chlorocebus aethiops (grivet monkey) / Cell: COS-1 / Plasmid: pcDNA3
Buffer solutionpH: 7
Buffer componentConc.: 20 mM / Name: HEPES / Formula: C8H18N2O4S
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 100 nm
Image recordingElectron dose: 37.2 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 2001

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
7UCSF ChimeraXmodel fitting
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
14Rosettamodel refinement
15Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 891373 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model

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