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- EMDB-40024: Locally refined cryoEM structure of receptor from beta-2-adrenerg... -

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Basic information

Entry
Database: EMDB / ID: EMD-40024
TitleLocally refined cryoEM structure of receptor from beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (transition intermediate #16 of 20)
Map dataSharpened 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
KeywordsGPCR / Adrenergic / Receptor / G protein / SIGNALING 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 / norepinephrine binding / Adrenoceptors / heat generation / positive regulation of autophagosome maturation / positive regulation of AMPA receptor 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 / norepinephrine binding / Adrenoceptors / heat generation / positive regulation of autophagosome maturation / positive regulation of AMPA receptor activity / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction / positive regulation of lipophagy / response to psychosocial stress / negative regulation of multicellular organism growth / adrenergic receptor signaling pathway / endosome to lysosome transport / diet induced thermogenesis / neuronal dense core vesicle / positive regulation of protein kinase A signaling / adenylate cyclase binding / smooth muscle contraction / potassium channel regulator activity / positive regulation of bone mineralization / brown fat cell differentiation / adenylate cyclase-activating adrenergic receptor signaling pathway / regulation of sodium ion transport / bone resorption / activation of adenylate cyclase activity / response to cold / receptor-mediated endocytosis / clathrin-coated endocytic vesicle membrane / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / positive regulation of protein serine/threonine kinase activity / 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 / lysosome / early endosome / cell surface receptor signaling pathway / receptor complex / 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 / membrane / identical protein binding / nucleus / plasma membrane
Similarity search - Function
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)
Similarity search - Domain/homology
Beta-2 adrenergic receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsPapasergi-Scott MM / Skiniotis G
Funding support United States, 2 items
OrganizationGrant numberCountry
Other government
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: 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
DepositionMar 8, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_40024.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.8677 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-2.0142725 - 2.7312095
Average (Standard dev.)-0.000020359119 (±0.01925912)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 284.6056 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40024_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Unsharpened map

Fileemd_40024_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map

Fileemd_40024_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map

Fileemd_40024_half_map_2.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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

EntireName: 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

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

SupramoleculeName: 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)

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Macromolecule #1: Beta-2 adrenergic receptor

MacromoleculeName: Beta-2 adrenergic receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.767242 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGQPGN GSAFLLAPNR SHAPDHDVEN LYFQGTQQRD EVWVVGMGIV MSLIVLAIVF GNVLVITAI AKFERLQTVT NYFITSLACA DLVMGLAVVP FGAAHILTKT WTFGNFWCEF WTSIDVLCVT ASIETLCVIA V DRYFAITS ...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

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

MacromoleculeName: (5R,6R)-6-(methylamino)-5,6,7,8-tetrahydronaphthalene-1,2,5-triol
type: ligand / ID: 2 / Number of copies: 1 / Formula: G1I
Molecular weightTheoretical: 209.242 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging.
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen 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..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
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 ...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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 19918625
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationSoftware - Name: cryoSPARC / Software - details: 3DVA
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: Local refinement of receptor / Number images used: 138568
FSC plot (resolution estimation)

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