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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


G protein-coupled receptor complex / opsin binding / absorption of visible light / photoreceptor inner segment membrane / 11-cis retinal binding / G protein-coupled photoreceptor activity / cellular response to light stimulus / phototransduction, visible light / G protein-coupled serotonin receptor binding / positive regulation of protein localization to cell cortex ...G protein-coupled receptor complex / opsin binding / absorption of visible light / photoreceptor inner segment membrane / 11-cis retinal binding / G protein-coupled photoreceptor activity / cellular response to light stimulus / phototransduction, visible light / G protein-coupled serotonin receptor binding / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / arrestin family protein binding / G-protein beta/gamma-subunit complex / cellular response to catecholamine stimulus / photoreceptor cell maintenance / cell cortex region / rhodopsin mediated signaling pathway / adenylate cyclase-activating dopamine receptor signaling pathway / outer membrane / G-protein beta-subunit binding / photoreceptor outer segment membrane / regulation of mitotic spindle organization / photoreceptor disc membrane / cellular response to forskolin / negative regulation of synaptic transmission / photoreceptor outer segment / G-protein beta/gamma-subunit complex binding / heterotrimeric G-protein complex / GTPase activating protein binding / G-protein alpha-subunit binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to light stimulus / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / phototransduction / cellular response to prostaglandin E stimulus / guanyl-nucleotide exchange factor activity / retina development in camera-type eye / visual perception / response to peptide hormone / G protein-coupled receptor binding / GDP binding / midbody / protein-chromophore linkage / lysosomal membrane / cell-cell junction / protein folding / cell cycle / GTPase activity / cell division / Golgi membrane / centrosome / G protein-coupled receptor signaling pathway / membrane raft / GTP binding / protein phosphorylation / magnesium ion binding / integral component of plasma membrane / zinc ion binding / extracellular exosome / membrane / integral component of membrane / identical protein binding / plasma membrane / nucleus / cytoplasm
Amino terminal of the G-protein receptor rhodopsin / WD40-repeat-containing domain / G protein-coupled receptor, rhodopsin-like / Rhodopsin / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / G-protein, beta subunit / WD40 repeat / Opsin / G-protein, gamma subunit ...Amino terminal of the G-protein receptor rhodopsin / WD40-repeat-containing domain / G protein-coupled receptor, rhodopsin-like / Rhodopsin / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / G-protein, beta subunit / WD40 repeat / Opsin / G-protein, gamma subunit / G protein alpha subunit, helical insertion / G-protein gamma-like domain / Guanine nucleotide-binding protein, beta subunit / GPCR, rhodopsin-like, 7TM / WD40/YVTN repeat-like-containing domain superfamily / Rhodopsin, N-terminal / GGL domain / G-protein beta WD-40 repeat / P-loop containing nucleoside triphosphate hydrolase / Visual pigments (opsins) retinal binding site / G-protein gamma-like domain superfamily / 7 transmembrane receptor (rhodopsin family) / WD40-repeat-containing domain superfamily / G-protein alpha subunit / WD40 repeat, conserved site / WD domain, G-beta repeat
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
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. / 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.
Validation Report
SummaryFull reportAbout validation report
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.
  • Download structure data
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


GlcNAc

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GlcNAc

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 Fab16COMPLEX1,2,3,4,5,60MULTIPLE SOURCES
2Guanine nucleotide-binding protein alpha subunitCOMPLEX11RECOMBINANT
3Guanine nucleotide-binding protein beta/gamma subunitCOMPLEX2,31NATURAL
4antibody FAB fragment Fab16COMPLEX4,51RECOMBINANT
5RhodopsinCOMPLEX61RECOMBINANT
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 / Details: The sample was monodisperse. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 / 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-ID3D fitting-ID
16FUF1
21GOT1
36QNK1

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