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- PDB-9epp: Cryo-EM Structure of Jumping Spider Rhodopsin-1 bound to a Giq he... -

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Basic information

Entry
Database: PDB / ID: 9epp
TitleCryo-EM Structure of Jumping Spider Rhodopsin-1 bound to a Giq heterotrimer
Components
  • 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(i) subunit alpha-1
  • Kumopsin1
KeywordsMEMBRANE PROTEIN / Opsin / GPCR / G protein / Signaling Complex
Function / homology
Function and homology information


photoreceptor activity / phototransduction / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase regulator activity / adenylate cyclase-inhibiting serotonin receptor signaling pathway ...photoreceptor activity / phototransduction / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase regulator activity / adenylate cyclase-inhibiting serotonin receptor signaling pathway / visual perception / cellular response to forskolin / regulation of mitotic spindle organization / Regulation of insulin secretion / positive regulation of cholesterol biosynthetic process / G protein-coupled receptor binding / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / G protein-coupled receptor activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to peptide hormone / G-protein beta/gamma-subunit complex binding / centriolar satellite / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GDP binding / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / G protein activity / GTPase binding / Ca2+ pathway / retina development in camera-type eye / midbody / cell cortex / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / Extra-nuclear estrogen signaling / cell population proliferation / ciliary basal body / G protein-coupled receptor signaling pathway / lysosomal membrane / cell division / GTPase activity / synapse / centrosome / protein-containing complex binding / GTP binding / nucleolus / magnesium ion binding / Golgi apparatus / signal transduction / extracellular exosome / nucleoplasm / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Opsin lateral eye type / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / G-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. ...Opsin lateral eye type / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / G-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / 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 / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / Kumopsin1 / 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(i) subunit alpha-1
Similarity search - Component
Biological speciesHasarius adansoni (spider)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsTejero, O. / Pamula, F. / Koyanagi, M. / Nagata, T. / Afanasyev, P. / Das, I. / Deupi, X. / Sheves, M. / Terakita, A. / Schertler, G.F.X. ...Tejero, O. / Pamula, F. / Koyanagi, M. / Nagata, T. / Afanasyev, P. / Das, I. / Deupi, X. / Sheves, M. / Terakita, A. / Schertler, G.F.X. / Rodrigues, M.J. / Tsai, C.-J.
Funding supportEuropean Union, Switzerland, 4items
OrganizationGrant numberCountry
European Research Council (ERC)951644European Union
H2020 Marie Curie Actions of the European Commission701647European Union
Swiss National Science Foundation192760 Switzerland
Swiss National Science Foundation18563 Switzerland
CitationJournal: Nat Commun / Year: 2024
Title: Active state structures of a bistable visual opsin bound to G proteins.
Authors: Oliver Tejero / Filip Pamula / Mitsumasa Koyanagi / Takashi Nagata / Pavel Afanasyev / Ishita Das / Xavier Deupi / Mordechai Sheves / Akihisa Terakita / Gebhard F X Schertler / Matthew J ...Authors: Oliver Tejero / Filip Pamula / Mitsumasa Koyanagi / Takashi Nagata / Pavel Afanasyev / Ishita Das / Xavier Deupi / Mordechai Sheves / Akihisa Terakita / Gebhard F X Schertler / Matthew J Rodrigues / Ching-Ju Tsai /
Abstract: Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light ...Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the G and G heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.
History
DepositionMar 19, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 23, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
R: Kumopsin1
A: Guanine nucleotide-binding protein G(i) subunit alpha-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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,8375
Polymers121,5274
Non-polymers3101
Water00
1


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

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Components

#1: Protein Kumopsin1


Mass: 37282.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hasarius adansoni (spider) / Gene: HaRh1 / Cell line (production host): HEK 293 GnTi- / Production host: Homo sapiens (human) / References: UniProt: B1B1U5
#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40324.980 Da / Num. of mol.: 1
Mutation: A31R,D193S,L194I,D337C,T340K,V342T,I344L,K345Q,D350E,G352N,F354V
Source method: isolated from a genetically manipulated source
Details: Human Gi/Jumping Spider Gq Chimera,Human Gi/Jumping Spider Gq Chimera
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37285.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 6633.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768
#5: Chemical ChemComp-A1H6M / 11,20-Ethanoretinal / (2E)-2-[3-[(1E,3E)-2-methyl-4-(2,6,6-trimethylcyclohexen-1-yl)buta-1,3-dienyl]cyclohex-2-en-1-ylidene]ethanal / Retinal 6.11


Mass: 310.473 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H30O / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Ternary complex of Jumping Spider Rhodopsin-1 with a chimeric Giq heterotrimerCOMPLEX#1-#40MULTIPLE SOURCES
2Jumping Spider Rhodopsin-1COMPLEX#11RECOMBINANT
3Human Gi/Jumping Spider Gq ChimeraCOMPLEX#21RECOMBINANTGuanine nucleotide-binding protein G(i) subunit alpha-1 including the following mutations: A31R, D193S, L194I, D337C, T340K, V342T, I344L, K345Q, D350E, G352N, F354V
4Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1COMPLEX#31RECOMBINANT
5Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Hasarius adansoni (spider)243517
33Hasarius adansoni (spider)243517
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Homo sapiens (human)9606HEK 293 GnTi-
33Trichoplusia ni (cabbage looper)7111High Five
44Trichoplusia ni (cabbage looper)7111High Five
55Trichoplusia ni (cabbage looper)7111High Five
Buffer solutionpH: 6.5
SpecimenConc.: 8.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159665 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0147641
ELECTRON MICROSCOPYf_angle_d1.31310373
ELECTRON MICROSCOPYf_dihedral_angle_d4.4561053
ELECTRON MICROSCOPYf_chiral_restr0.0451177
ELECTRON MICROSCOPYf_plane_restr0.0041302

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