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- PDB-6qno: Rhodopsin-Gi protein complex -

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Basic information

Entry
Database: PDB / ID: 6qno
TitleRhodopsin-Gi protein complex
Components
  • (Fab antibody fragment ...) x 2
  • (Guanine nucleotide-binding protein ...) x 3
  • Rhodopsin
KeywordsSIGNALING PROTEIN / GPCR and G protein 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 / 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) ...Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / 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 / 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 / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / G-protein alpha-subunit binding / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / sperm midpiece / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / visual perception / guanyl-nucleotide exchange factor activity / Regulation of insulin secretion / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / protein localization / ADP signalling through P2Y purinoceptor 12 / response to peptide hormone / microtubule cytoskeleton organization / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / sensory perception of taste / GPER1 signaling / 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 cortex / midbody / G alpha (i) signalling events / G alpha (s) signalling events / cell population proliferation
Similarity search - Function
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. / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit ...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. / G-protein alpha subunit, group I / 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) / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RETINAL / Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Rhodopsin / 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 speciesHomo sapiens (human)
Mus musculus (house mouse)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.38 Å
AuthorsTsai, C.-J. / Marino, J. / Adaixo, R.J. / Pamula, F. / Muehle, J. / Maeda, S. / Flock, T. / Taylor, N.M.I. / Mohammed, I. / Matile, H. ...Tsai, C.-J. / Marino, J. / Adaixo, R.J. / Pamula, F. / Muehle, J. / Maeda, S. / Flock, T. / Taylor, N.M.I. / Mohammed, I. / Matile, H. / Dawson, R.J.P. / Deupi, X. / Stahlberg, H. / Schertler, G.F.X.
Funding support Switzerland, 5items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_153145 Switzerland
Swiss National Science Foundation310030B_173335 Switzerland
Swiss National Science Foundation160805 Switzerland
Swiss Nanoscience InstituteA13.12 NanoGhip Switzerland
Swiss National Science FoundationNCCR TransCure Switzerland
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.
Authors: Ching-Ju Tsai / Jacopo Marino / Ricardo Adaixo / Filip Pamula / Jonas Muehle / Shoji Maeda / Tilman Flock / Nicholas Mi Taylor / Inayatulla Mohammed / Hugues Matile / Roger Jp Dawson / ...Authors: Ching-Ju Tsai / Jacopo Marino / Ricardo Adaixo / Filip Pamula / Jonas Muehle / Shoji Maeda / Tilman Flock / Nicholas Mi Taylor / Inayatulla Mohammed / Hugues Matile / Roger Jp Dawson / Xavier Deupi / Henning Stahlberg / Gebhard Schertler /
Abstract: One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently ...One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
History
DepositionFeb 11, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / entity / entity_src_gen / entity_src_nat / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _em_entity_assembly.entity_id_list / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _entity_src_gen.gene_src_common_name / _entity_src_nat.common_name / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4598
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
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(T) subunit gamma-T1
L: Fab antibody fragment light chain
H: Fab antibody fragment heavy chain
R: Rhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)181,7748
Polymers181,0656
Non-polymers7092
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The components of the complex were mixed., followed by gel filtration. The peak sample contains all the protein components, as evaluated by SDS-PAGE.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14070 Å2
ΔGint-70 kcal/mol
Surface area61180 Å2
MethodPISA

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#1: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 43182.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P63096
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37416.930 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Tissue: retina / References: UniProt: P62871
#3: Protein Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Transducin gamma chain


Mass: 8556.918 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Tissue: retina / References: UniProt: P02698

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Antibody , 2 types, 2 molecules LH

#4: Antibody Fab antibody fragment light chain


Mass: 26309.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): hybridoma cell / Production host: hybrid (others)
#5: Antibody Fab antibody fragment heavy chain


Mass: 26558.902 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): hybridoma cell / Production host: hybrid (others)

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Protein / Sugars / Non-polymers , 3 types, 3 molecules R

#6: Protein Rhodopsin /


Mass: 39040.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: mutant N2C/M257Y/D282C / Source: (gene. exp.) Bos taurus (cattle) / Tissue: retina / Gene: RHO / Cell line (production host): HEK293 GnTI- / Production host: Homo sapiens (human) / References: UniProt: P02699
#7: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#8: 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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Details

Has ligand of interestY

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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-Gi complex bound with antibody fragment Fab16COMPLEX#1-#60MULTIPLE SOURCES
2Guanine nucleotide-binding protein alpha subunitCOMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein beta/gamma subunitCOMPLEX#2-#31NATURAL
4antibody FAB fragment Fab16COMPLEX#4-#51RECOMBINANT
5RhodopsinCOMPLEX#61RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Bos taurus (cattle)9913
34Mus musculus (house mouse)10090
45Bos taurus (cattle)9913
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21 (bacteria)511693
34hybrid (others)37965
45Homo sapiens (human)9606
Buffer solutionpH: 7.5
Details: The detergent lauryl-maltose neopentyl glycol (LMNG) was used before the last purification step by gel filtration. In the gel filtration, detergent-free buffer was used.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium cholorideNaClSodium chloride1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K

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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 / Nominal magnification: 165000 X / Calibrated magnification: 165000 X / Nominal defocus max: 25000 nm / Nominal defocus min: 15000 nm / Calibrated defocus min: 15000 nm / Calibrated defocus max: 25000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 70 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3200
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimera1.11.2model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13-2998model refinement
14Coot0.8.9model refinement
Image processingDetails: super resolution mode
CTF correctionType: NONE
Particle selectionNum. of particles selected: 580000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115000 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16FUF

6fuf

1
21GOT

1got

1
36QNK

6qnk

1

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