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- PDB-6oya: Structure of the Rhodopsin-Transducin-Nanobody Complex -

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Basic information

Entry
Database: PDB / ID: 6oya
TitleStructure of the Rhodopsin-Transducin-Nanobody Complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Camelid antibody VHH fragment
  • Gt-alpha/Gi1-alpha chimera
  • Rhodopsin
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / GPCR / G protein / Complex / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / negative regulation of cyclic-nucleotide phosphodiesterase activity / podosome assembly / Olfactory Signaling Pathway / absorption of visible light / Sensory perception of sweet, bitter, and umami (glutamate) taste ...Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / negative regulation of cyclic-nucleotide phosphodiesterase activity / podosome assembly / Olfactory Signaling Pathway / absorption of visible light / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / opsin binding / The canonical retinoid cycle in rods (twilight vision) / : / G protein-coupled photoreceptor activity / photoreceptor inner segment membrane / rhodopsin mediated signaling pathway / 11-cis retinal binding / eye photoreceptor cell development / cellular response to light stimulus / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / phototransduction, visible light / thermotaxis / Activation of the phototransduction cascade / detection of temperature stimulus involved in thermoception / outer membrane / arrestin family protein binding / photoreceptor cell maintenance / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / Adrenaline,noradrenaline inhibits insulin secretion / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Glucagon-type ligand receptors / acyl binding / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / Extra-nuclear estrogen signaling / G alpha (z) signalling events / G alpha (s) signalling events / G alpha (q) signalling events / photoreceptor outer segment membrane / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / response to light stimulus / phototransduction / photoreceptor outer segment / G-protein alpha-subunit binding / sperm midpiece / visual perception / photoreceptor inner segment / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / protein localization / microtubule cytoskeleton organization / photoreceptor disc membrane / cellular response to catecholamine stimulus / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / sensory perception of taste / GDP binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / cell-cell junction / signaling receptor complex adaptor activity / retina development in camera-type eye / GTPase binding / gene expression / phospholipase C-activating G protein-coupled receptor signaling pathway / cell population proliferation / G protein-coupled receptor signaling pathway / Golgi membrane / GTPase activity / protein-containing complex binding / GTP binding / protein kinase binding / zinc ion binding / membrane / identical protein binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins ...Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / YVTN repeat-like/Quinoprotein amine dehydrogenase / G-protein alpha subunit, group I / 7 Propeller / Methylamine Dehydrogenase; Chain H / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / 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) / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
RETINAL / Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Rhodopsin / Guanine nucleotide-binding protein G(t) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Similarity search - Component
Biological speciesBos taurus (cattle)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGao, Y. / Hu, H. / Ramachandran, S. / Erickson, J.W. / Cerione, R.A. / Skiniotis, G.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA201402 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122575 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS092695 United States
CitationJournal: Mol Cell / Year: 2019
Title: Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.
Authors: Yang Gao / Hongli Hu / Sekar Ramachandran / Jon W Erickson / Richard A Cerione / Georgios Skiniotis /
Abstract: Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (G) by catalyzing GDP-GTP exchange on its α subunit (Gα). To elucidate the ...Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (G) by catalyzing GDP-GTP exchange on its α subunit (Gα). To elucidate the determinants of G coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-G complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how G overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the Gα helical domain (αHD) contacting the Gβγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gβγ during receptor-catalyzed nucleotide exchange.
History
DepositionMay 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Nov 27, 2019Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Gt-alpha/Gi1-alpha chimera
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(T) subunit gamma-T1
N: Camelid antibody VHH fragment
R: Rhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,3136
Polymers142,0295
Non-polymers2841
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12830 Å2
ΔGint-68 kcal/mol
Surface area45240 Å2

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Components

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Protein , 2 types, 2 molecules AR

#1: Protein Gt-alpha/Gi1-alpha chimera


Mass: 41175.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Production host: Escherichia coli (E. coli) / References: UniProt: P04695
#5: Protein Rhodopsin /


Mass: 39031.457 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P02699

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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: 37430.957 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P62871
#3: Protein Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Transducin gamma chain


Mass: 9337.784 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P02698

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

#4: Antibody Camelid antibody VHH fragment


Mass: 15052.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#6: Chemical ChemComp-RET / RETINAL / Retinal


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

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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-IDSource
1Rhodopsin-Transducin-Nanobody ComplexCOMPLEX#1-#50MULTIPLE SOURCES
2TransducinCOMPLEX#1-#31RECOMBINANT
3RhodopsinCOMPLEX#51NATURAL
4NanobodySingle-domain antibodyCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.1418 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Bos taurus (cattle)9913
23Bos taurus (cattle)9913
34Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
24Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %
Details: Blot for 1 second before plunging; avoid light as much as possible.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3837
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.06CTF correction
12RELIONclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 309116 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE

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