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- PDB-8s2t: Cryo-EM structure of cell-free synthesized Human beta-1 adrenergi... -

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Basic information

Entry
Database: PDB / ID: 8s2t
TitleCryo-EM structure of cell-free synthesized Human beta-1 adrenergic receptor cotranslationally inserted into a lipidic nanodiscs
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Beta-1 adrenergic receptor
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Nanobody-35
KeywordsMEMBRANE PROTEIN / Signal transduction / GPCR / co-translational nanodisc insertion / G-protein
Function / homology
Function and homology information


positive regulation of heart rate by epinephrine-norepinephrine / positive regulation of the force of heart contraction by epinephrine-norepinephrine / beta-adrenergic receptor activity / beta1-adrenergic receptor activity / G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / alpha-2A adrenergic receptor binding / regulation of circadian sleep/wake cycle, sleep / fear response / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation ...positive regulation of heart rate by epinephrine-norepinephrine / positive regulation of the force of heart contraction by epinephrine-norepinephrine / beta-adrenergic receptor activity / beta1-adrenergic receptor activity / G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / alpha-2A adrenergic receptor binding / regulation of circadian sleep/wake cycle, sleep / fear response / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / heat generation / Adrenoceptors / negative regulation of multicellular organism growth / diet induced thermogenesis / neuronal dense core vesicle / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / brown fat cell differentiation / renal water homeostasis / Hedgehog 'off' state / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / response to cold / trans-Golgi network membrane / PDZ domain binding / negative regulation of inflammatory response to antigenic stimulus / Schaffer collateral - CA1 synapse / bone development / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled 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 / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / 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 / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of cold-induced thermogenesis / G protein activity / GTPase binding / Ca2+ pathway / retina development in camera-type eye / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation / G alpha (i) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / Ras protein signal transduction / early endosome / Extra-nuclear estrogen signaling / cell population proliferation / positive regulation of MAPK cascade / G protein-coupled receptor signaling pathway / protein heterodimerization activity / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding
Similarity search - Function
Beta 1 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit ...Beta 1 adrenoceptor / Adrenoceptor family / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / 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. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ISOPRENALINE / Beta-1 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 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMerino, F. / Koeck, Z. / Ermel, U. / Dahlhaus, P. / Doetsch, V. / Kubicek, J. / Frangakis, A.S. / Hilger, D. / Bernhard, F.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)BE1911/9-1 Germany
German Research Foundation (DFG)FR 1653/12-2 Germany
CitationJournal: To Be Published
Title: Cryo-EM structure of cell-free synthesized Human beta-1 adrenergic receptor cotranslationally inserted into a lipidic nanodiscs
Authors: Merino, F. / Koeck, Z. / Ermel, U. / Dahlhaus, P. / Doetsch, V. / Frangakis, A.S. / Hilger, D. / Bernhard, F.
History
DepositionFeb 19, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0May 21, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
N: Nanobody-35
R: Beta-1 adrenergic receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,6746
Polymers155,4635
Non-polymers2111
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13240 Å2
ΔGint-60 kcal/mol
Surface area41570 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AR

#1: Protein Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha 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
#5: Protein Beta-1 adrenergic receptor / Beta-1 adrenoreceptor / Beta-1 adrenoceptor


Mass: 51130.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cell-free synthesized / Source: (gene. exp.) Homo sapiens (human) / Gene: ADRB1, ADRB1R, B1AR / Production host: Escherichia coli (E. coli) / References: UniProt: P08588

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules BC

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37728.152 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 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7563.750 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

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Antibody / Non-polymers , 2 types, 2 molecules N

#4: Antibody Nanobody-35


Mass: 14714.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#6: Chemical ChemComp-5FW / ISOPRENALINE


Mass: 211.258 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H17NO3 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM

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Details

Has ligand of interestY
Has protein modificationY

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Complex between, human Beta-1 adrenergic receptor, Gs, and nanobody-35COMPLEXThe GPCR was produced cell-free and co-translationally inserted into a lipidic nanodisc. Additional Gs and Nanobody-35 during GPCR production leads to the full assembly of the complex.#1-#3, #5, #40RECOMBINANT
2Heterotrimeric Gs complexCOMPLEXProduced in T. ni cells for later in vitro assembly of full complex.#1-#31RECOMBINANT
3Beta-1 adrenergic receptorCOMPLEXcell-free synthesized gpcr co-translationally inserted into a nanodisc#51RECOMBINANT
4Nanobody-35COMPLEXNanobody that binds to Galpha-Gbeta#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.155 MDaNO
210.089 MDaNO
310.051 MDaNO
410.015 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Trichoplusia ni (cabbage looper)7111
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 65 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum SE / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategoryDetails
1Topazparticle selection
4CTFFINDCTF correction
7UCSF ChimeraXmodel fitting
9Cootmodel refinementManual corrections
10Rosettamodel refinementIterative rebuild plus relaxation
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
13RELIONclassification
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 235380
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74266 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
16E3Y

6e3y

6E3YUsed for Gs and Nb351PDBexperimental model
2USed for beta-1 ARAlphaFoldin silico model

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