EMDB-40028: Locally refined cryoEM structure of receptor from beta-2-adrenerg...

Basic information

Entry

Database: EMDB / ID: EMD-40028

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

Sample

• Complex: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP
  • Protein or peptide: Beta-2 adrenergic receptor
• Ligand: (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol

• 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 / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / cellular response to amyloid-beta / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / positive regulation of cold-induced thermogenesis / amyloid-beta binding / G alpha (s) signalling events / transcription by RNA polymerase II / positive regulation of MAPK cascade / early endosome / lysosome / receptor complex / cell surface receptor signaling pathway / endosome membrane / Ub-specific processing proteases / endosome / apical plasma membrane / protein-containing complex binding / Golgi apparatus / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / identical protein binding / membrane / nucleus / plasma membrane

Domain/homology:

Beta 2 adrenoceptor / Adrenoceptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / 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)

Component:

Beta-2 adrenergic receptor

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 4.1 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 8, 2023	-
Header (metadata) release	Mar 6, 2024	-
Map release	Mar 6, 2024	-
Update	Nov 13, 2024	-
Current status	Nov 13, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_40028.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Sharpened map
• Voxel size: X=Y=Z: 0.8677 Å

Density

Contour Level	By AUTHOR: 0.12
Minimum - Maximum	-0.7965496 - 1.3187189
Average (Standard dev.)	0.00003681887 (±0.015281114)

• 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_40028_msk_1.map

Additional map: Unsharpened map

• File: emd_40028_additional_1.map
• Annotation: Unsharpened map

Half map: Half map

• File: emd_40028_half_map_1.map
• Annotation: Half map

Half map: Half map

• File: emd_40028_half_map_2.map
• Annotation: Half 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: Beta-2 adrenergic receptor
• Ligand: (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol

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: #1; 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: Beta-2 adrenergic receptor

• Macromolecule: Name: Beta-2 adrenergic receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 51.767242 KDa
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)

Sequence

String:

MKTIIALSYI FCLVFADYKD DDDAMGQPGN GSAFLLAPNR SHAPDHDVEN LYFQGTQQRD EVWVVGMGIV MSLIVLAIVF GNVLVITAI AKFERLQTVT NYFITSLACA DLVMGLAVVP FGAAHILTKT WTFGNFWCEF WTSIDVLCVT ASIETLCVIA V DRYFAITS PFKYQSLLTK NKARVIILMV WIVSGLTSFL PIQMHWYRAT HQEAINCYAE ETCCDFFTNQ AYAIASSIVS FY VPLVIMV FVYSRVFQEA KRQLQKIDKS EGRFHVQNLS QVEQDGRTGH GLRRSSKFCL KEHKALKTLG IIMGTFTLCW LPF FIVNIV HVIQDNLIRK EVYILLNWIG YVNSGFNPLI YCRSPDFRIA FQELLCLRRS SLKAYGNGYS SNGNTGEQSG LEVL FQGPY HVEQEKENKL LAEDLPGTED FVGHQGTVPS DNIDSQGRNA STNDSLLETS QVAPA

UniProtKB: Beta-2 adrenergic receptor

Macromolecule #2: (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol

• Macromolecule: Name: (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol; type: ligand / ID: 2 / Number of copies: 1 / Formula: G1I
• Molecular weight: Theoretical: 209.242 Da

Chemical component information

ChemComp-G1I:
(5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol

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.
• Grid: Model: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / 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) / Detector mode: COUNTING / 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: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Local refinement of receptor / Number images used: 47951
• Initial angle assignment: Type: ANGULAR RECONSTITUTION
• Final angle assignment: Type: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
• Final 3D classification: Software - Name: cryoSPARC / Software - details: 3DVA