EMDB-40176: Locally refined cryoEM structure of G protein heterotrimer from b...

Basic information

Entry

Database: EMDB / ID: EMD-40176

• Title: Locally refined cryoEM structure of G protein heterotrimer from beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (transition intermediate #20 of 20)

Sample

• Complex: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP
  • Protein or peptide: G protein alpha s (short)
  • Protein or peptide: G protein beta 1
  • Protein or peptide: G protein gamma 2
  • Protein or peptide: Beta-2 adrenergic receptor

• Keywords: GPCR / Adrenergic / Receptor / G protein / SIGNALING PROTEIN

Function / homology

Function:

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 / adenylate cyclase binding / smooth muscle contraction / potassium channel regulator activity / bone resorption / positive regulation of bone mineralization / brown fat cell differentiation / adenylate cyclase-activating adrenergic receptor signaling pathway / regulation of sodium ion transport / response to cold / receptor-mediated endocytosis / clathrin-coated endocytic vesicle membrane / positive regulation of protein serine/threonine kinase activity / Olfactory Signaling Pathway / Activation of the phototransduction cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / 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 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / 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 / cellular response to amyloid-beta / adenylate cyclase-activating dopamine receptor signaling pathway / 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 / 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 / positive regulation of cold-induced thermogenesis / retina development in camera-type eye / amyloid-beta binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / fibroblast proliferation / G alpha (i) signalling events / G alpha (s) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (q) signalling events / Ras protein signal transduction / transcription by RNA polymerase II / cell population proliferation / positive regulation of MAPK cascade / Extra-nuclear estrogen signaling / early endosome / lysosome / receptor complex / cell surface receptor signaling pathway / endosome membrane / Ub-specific processing proteases / endosome / G protein-coupled receptor signaling pathway / apical plasma membrane / lysosomal membrane

Domain/homology:

Beta 2 adrenoceptor / Adrenoceptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / 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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily

Component:

Beta-2 adrenergic receptor / 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 / Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.4 Å
• Authors: Papasergi-Scott MM / Skiniotis G

Funding support

United States, 2 items

Organization	Grant number	Country
Other government
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)		United States

• 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 / 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 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

Deposition	Mar 16, 2023	-
Header (metadata) release	Mar 6, 2024	-
Map release	Mar 6, 2024	-
Update	Jun 12, 2024	-
Current status	Jun 12, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_40176.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.8677 Å

Density

Contour Level	By AUTHOR: 0.2
Minimum - Maximum	-0.5769803 - 0.9892217
Average (Standard dev.)	-0.00002857563 (±0.020649865)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 328 328 328 Spacing 328 328 328
Cell	A=B=C: 284.6056 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_40176_msk_1.map

Additional map: #1

• File: emd_40176_additional_1.map

Half map: #1

• File: emd_40176_half_map_1.map

Half map: #2

• File: emd_40176_half_map_2.map

Sample components

Entire : Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP

• Entire: Name: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP

Components

• Complex: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP
  • Protein or peptide: G protein alpha s (short)
  • Protein or peptide: G protein beta 1
  • Protein or peptide: G protein gamma 2
  • Protein or peptide: Beta-2 adrenergic receptor

Supramolecule #1: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP

• Supramolecule: Name: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP; type: complex / ID: 1 / Parent: 0 / Macromolecule list: all; Details: Combined datasets of the protein complex from samples plunge frozen at 5 sec, 10 sec, or 17 sec after GTP addition to the sample.
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: G protein alpha s (short)

• Macromolecule: Name: G protein alpha s (short) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO

Sequence

String:

MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG DSEKATKVQD IKNNLKEAIE TIVAAMSNLV PPVELANPEN QFRVDYILSV MNVPDFDFPP EFYEHAKALW EDEGVRACYE RSNEYQLIDC AQYFLDKIDV IKQADYVPSD QDLLRCRVLT SGIFETKFQV DKVNFHMFDV GGQRDERRKW IQCFNDVTAI IFVVASSSYN MVIREDNQTN RLQEALNLFK SIWNNRWLRT ISVILFLNKQ DLLAEKVLAG KSKIEDYFPE FARYTTPEDA TPEPGEDPRV TRAKYFIRDE FLRISTASGD GRHYCYPHFT CAVDTENIRR VFNDCRDIIQ RMHLRQYELL

UniProtKB: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

Macromolecule #2: G protein beta 1

• Macromolecule: Name: G protein beta 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO

Sequence

String:

GSSGSELDQL RQEAEQLKNQ IRDARKACAD ATLSQITNNI DPVGRIQMRT RRTLRGHLAK IYAMHWGTDS RLLVSASQDG KLIIWDSYTT NKVHAIPLRS SWVMTCAYAP SGNYVACGGL DNICSIYNLK TREGNVRVSR ELAGHTGYLS CCRFLDDNQI VTSSGDTTCA LWDIETGQQT TTFTGHTGDV MSLSLAPDTR LFVSGACDAS AKLWDVREGM CRQTFTGHES DINAICFFPN GNAFATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAGHDNRVSC LGVTDDGMAV ATGSWDSFLK IWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

Macromolecule #3: G protein gamma 2

• Macromolecule: Name: G protein gamma 2 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO

Sequence

String:

MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

Macromolecule #4: Beta-2 adrenergic receptor

• Macromolecule: Name: Beta-2 adrenergic receptor / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO

Sequence

String:

MKTIIALSYI FCLVFADYKD DDDAMGQPGN GSAFLLAPNR SHAPDHDVEN LYFQGTQQRD EVWVVGMGIV MSLIVLAIVF GNVLVITAIA KFERLQTVTN YFITSLACAD LVMGLAVVPF GAAHILTKTW TFGNFWCEFW TSIDVLCVTA SIETLCVIAV DRYFAITSPF KYQSLLTKNK ARVIILMVWI VSGLTSFLPI QMHWYRATHQ EAINCYAEET CCDFFTNQAY AIASSIVSFY VPLVIMVFVY SRVFQEAKRQ LQKIDKSEGR FHVQNLSQVE QDGRTGHGLR RSSKFCLKEH KALKTLGIIM GTFTLCWLPF FIVNIVHVIQ DNLIRKEVYI LLNWIGYVNS GFNPLIYCRS PDFRIAFQEL LCLRRSSLKA YGNGYSSNGN TGEQSGLEVL FQGPYHVEQE KENKLLAEDL PGTEDFVGHQ GTVPSDNIDS QGRNASTNDS LLETSQVAPA

UniProtKB: Beta-2 adrenergic receptor

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5 ; Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging.
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV; Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging..

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Ų; Details: The map 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.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 19918625
• Startup model: Type of model: INSILICO MODEL
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC; Software - details: Local refinement of G protein heterotrimer; Number images used: 47951
• Initial angle assignment: Type: ANGULAR RECONSTITUTION
• Final angle assignment: Type: ANGULAR RECONSTITUTION