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Yorodumi- PDB-8gfw: CryoEM structure of beta-2-adrenergic receptor in complex with GT... -
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-Basic information
Entry | Database: PDB / ID: 8gfw | |||||||||
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Title | CryoEM structure of beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (transition intermediate #2 of 20) | |||||||||
Components |
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Keywords | SIGNALING PROTEIN / GPCR / Adrenergic / Receptor / G protein | |||||||||
Function / homology | Function and homology information beta2-adrenergic receptor activity / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / positive regulation of mini excitatory postsynaptic potential / positive regulation of cAMP-dependent protein kinase activity / positive regulation of AMPA receptor activity / norepinephrine binding / positive regulation of autophagosome maturation / heat generation / Adrenoceptors / activation of transmembrane receptor protein tyrosine kinase activity ...beta2-adrenergic receptor activity / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / positive regulation of mini excitatory postsynaptic potential / positive regulation of cAMP-dependent protein kinase activity / positive regulation of AMPA receptor activity / norepinephrine binding / positive regulation of autophagosome maturation / heat generation / Adrenoceptors / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction / positive regulation of lipophagy / negative regulation of multicellular organism growth / negative regulation of G protein-coupled receptor signaling pathway / response to psychosocial stress / endosome to lysosome transport / adrenergic receptor signaling pathway / diet induced thermogenesis / positive regulation of protein kinase A signaling / neuronal dense core vesicle / PKA activation in glucagon signalling / hair follicle placode formation / adenylate cyclase binding / developmental growth / smooth muscle contraction / D1 dopamine receptor binding / intracellular transport / renal water homeostasis / Hedgehog 'off' state / potassium channel regulator activity / bone resorption / positive regulation of bone mineralization / brown fat cell differentiation / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / regulation of sodium ion transport / cellular response to glucagon stimulus / adenylate cyclase activator activity / regulation of insulin secretion / response to cold / receptor-mediated endocytosis / trans-Golgi network membrane / negative regulation of inflammatory response to antigenic stimulus / clathrin-coated endocytic vesicle membrane / bone development / positive regulation of protein serine/threonine kinase activity / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G protein activity / 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 / platelet aggregation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / cognition / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to amyloid-beta / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / cellular response to prostaglandin E stimulus / Inactivation, recovery and regulation of the phototransduction cascade / G-protein beta-subunit binding / heterotrimeric G-protein complex / sensory perception of smell / G alpha (12/13) signalling events / extracellular vesicle / Cargo recognition for clathrin-mediated endocytosis / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / GTPase binding / Clathrin-mediated endocytosis / retina development in camera-type eye / positive regulation of cold-induced thermogenesis / amyloid-beta binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | |||||||||
Authors | Papasergi-Scott, M.M. / Skiniotis, G. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nature / Year: 2024 Title: Time-resolved cryo-EM of G-protein activation by a GPCR. Authors: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul ...Authors: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul / Peter Gmeiner / Brian K Kobilka / Peter W Hildebrand / Georgios Skiniotis / Abstract: G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM ...G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G protein in complex with the β-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8gfw.cif.gz | 196.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8gfw.ent.gz | 142.7 KB | Display | PDB format |
PDBx/mmJSON format | 8gfw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8gfw_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8gfw_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8gfw_validation.xml.gz | 41.3 KB | Display | |
Data in CIF | 8gfw_validation.cif.gz | 61 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/gf/8gfw ftp://data.pdbj.org/pub/pdb/validation_reports/gf/8gfw | HTTPS FTP |
-Related structure data
Related structure data | 29986MC 8gdzC 8ge1C 8ge2C 8ge3C 8ge4C 8ge5C 8ge6C 8ge7C 8ge8C 8ge9C 8geaC 8gebC 8gecC 8gedC 8geeC 8gefC 8gegC 8gehC 8geiC 8gejC 8gfvC 8gfxC 8gfyC 8gfzC 8gg0C 8gg1C 8gg2C 8gg3C 8gg4C 8gg5C 8gg6C 8gg7C 8gg8C 8gg9C 8ggaC 8ggbC 8ggcC 8ggeC 8ggfC 8ggiC 8ggjC 8ggkC 8gglC 8ggmC 8ggnC 8ggoC 8ggpC 8ggqC 8ggrC 8ggsC 8ggtC 8gguC 8ggvC 8ggwC 8ggxC 8ggyC 8ggzC 8gh0C 8gh1C 8unlC 8unmC 8unnC 8unoC 8unpC 8unqC 8unrC 8unsC 8untC 8unuC 8unvC 8unwC 8unxC 8unyC 8unzC 8uo0C 8uo1C 8uo2C 8uo3C 8uo4C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG
#1: Protein | Mass: 44326.160 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092 |
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#2: Protein | Mass: 37573.988 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 |
#3: Protein | Mass: 7861.143 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 |
-Protein , 1 types, 1 molecules R
#4: Protein | Mass: 51767.242 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ADRB2, ADRB2R, B2AR / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P07550 |
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-Non-polymers , 3 types, 7 molecules
#5: Chemical | ChemComp-GTP / |
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#6: Chemical | ChemComp-G1I / ( |
#7: Water | ChemComp-HOH / |
-Details
Has ligand of interest | N |
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Has protein modification | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of beta-2 adrenergic receptor and GTP-bound Gs heterotrimer Type: COMPLEX Details: Combined datasets of complex plunge frozen at 5 sec, 10 sec, or 17 sec after GTP addition to the sample. Entity ID: #1-#4 / Source: MULTIPLE SOURCES | ||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||
Source (recombinant) |
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Buffer solution | pH: 7.5 Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging. | ||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Specimen support | Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) Details: The Structure is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 ...Details: The Structure is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 Summit direct electron detector (Gatan). The microscope was operated at 300 kV accelerating voltage, with a nominal magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. A total exposure of 60.48 electrons/ Angstrom^2 over 63 frames with defocus ranging from -1.0 - -2.0 micrometers was used. Cryo-EM imaging of beta-2AR-Gs + GTP (10 sec) complex was performed on four separate grids over three collection sessions. The microscope was operated at 300 kV accelerating voltage, with a magnification at the camera of 58,679x in counting mode resulting in a magnified pixel size of 0.8521 Angstrom. For the first and second grids, movies were obtained at an exposure rate of 21.13 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For an additional collection of the first grid, movies were obtained at an exposure rate of 20.95 electrons/ Angstrum^2/sec with defocus ranging from -0.4 -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For the third and fourth grids, movies were obtained at an exposure rate of 30.71 electrons/ Angstrum^2/sec with defocus ranging from -0.5 - -1.6 micrometers. The total exposure time was 2.008 sec over 79 frames per movie stack. Cryo-EM imaging of beta-2AR-Gs + GTP (17 sec) was performed on a Titan Krios equipped with a post-column energy filter, with a magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. Movies were obtained at an exposure rate of 32.46 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -0.9 micrometers. The total exposure time was 1.999 sec over 79 frames per movie stack. |
-Processing
EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 19918625 | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73129 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Refine LS restraints |
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