9YGZ
Cryo-EM structure of active mutant human green cone opsin (E129Q) in complex with chimeric G protein (miniGist)
Summary for 9YGZ
| Entry DOI | 10.2210/pdb9ygz/pdb |
| EMDB information | 72946 |
| Descriptor | Medium-wave-sensitive opsin 1, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2, ... (6 entities in total) |
| Functional Keywords | g protein-coupled receptor, cone opsin, signaling protein, membrane protein |
| Biological source | Homo sapiens (human) More |
| Total number of polymer chains | 5 |
| Total formula weight | 173863.98 |
| Authors | |
| Primary citation | Yao, W.,Fay, J.F.,Farrens, D.L. Biophysical and structural analysis of human green cone opsin. Biophys.J., 2026 Cited by PubMed 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 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. PubMed: 41832604DOI: 10.1016/j.bpj.2026.03.029 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (3.04 Å) |
Structure validation
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