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- PDB-9rx1: Cryo-EM structure of a single-chain beta1-adrenoceptor - AmpC bet... -

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Basic information

Entry
Database: PDB / ID: 9rx1
TitleCryo-EM structure of a single-chain beta1-adrenoceptor - AmpC beta-lactamase fusion protein
ComponentsBeta-1 adrenergic receptor,Beta-lactamase
KeywordsSIGNALING PROTEIN / G protein-coupled receptor / cyanopindolol / AmpC beta-lactamase / fusion protein / cryo-EM
Function / homology
Function and homology information


beta1-adrenergic receptor activity / positive regulation of heart contraction / regulation of circadian sleep/wake cycle, sleep / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / antibiotic catabolic process / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of cardiac muscle cell apoptotic process / beta-lactamase / beta-lactamase activity / outer membrane-bounded periplasmic space ...beta1-adrenergic receptor activity / positive regulation of heart contraction / regulation of circadian sleep/wake cycle, sleep / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / antibiotic catabolic process / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of cardiac muscle cell apoptotic process / beta-lactamase / beta-lactamase activity / outer membrane-bounded periplasmic space / early endosome / positive regulation of MAPK cascade / response to antibiotic / membrane / identical protein binding / plasma membrane
Similarity search - Function
Beta 1 adrenoceptor / : / : / Beta-lactamase, class-C active site / Beta-lactamase class-C active site. / : / Beta-lactamase-related / Beta-lactamase / Adrenoceptor family / Serpentine type 7TM GPCR chemoreceptor Srsx ...Beta 1 adrenoceptor / : / : / Beta-lactamase, class-C active site / Beta-lactamase class-C active site. / : / Beta-lactamase-related / Beta-lactamase / Adrenoceptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / Beta-lactamase/transpeptidase-like / G-protein coupled receptors family 1 signature. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile.
Similarity search - Domain/homology
Chem-P32 / Beta-lactamase / Beta-1 adrenergic receptor
Similarity search - Component
Biological speciesMeleagris gallopavo (turkey)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBenoit, R.M. / Afanasyev, P.
Funding support Switzerland, 3items
OrganizationGrant numberCountry
Novartis FreeNovation
Promedica Siftung1401/M Switzerland
Swiss National Science FoundationCRSK-3_190414 Switzerland
CitationJournal: J Struct Biol / Year: 2026
Title: Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein.
Authors: Gabriella Collu / Inayathulla Mohammed / Aleix Lafita / Tobias Bierig / Emiliya Poghosyan / Spencer Bliven / Julius Rabl / Pavel Afanasyev / Roger M Benoit /
Abstract: The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We ...The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC β-lactamase into the third intracellular loop (ICL3) of ultra-thermostable β1-adrenoceptor (β1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known β1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 Å. The seven-helix architecture and helix eight, as well as both GPCR - AmpC β-lactamase connections are clearly resolved. β1AR is in an inactive-like conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices. The map contains clear density for the cyanopindolol. The fusion protein geometry is expected to be compatible with a subset of other class A GPCRs exhibiting suitable architecture. For receptors meeting these geometric requirements, this approach may facilitate cryo-EM structure determination of GPCR-ligand complexes in an inactive-like state. In addition, it could support structural studies of GPCRs in the absence of ligands.
History
DepositionJul 10, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 22, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-1 adrenergic receptor,Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,2712
Polymers72,9841
Non-polymers2871
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Beta-1 adrenergic receptor,Beta-lactamase / Beta-1 adrenoreceptor / Beta-1 adrenoceptor / Beta-T / Cephalosporinase / CSase


Mass: 72983.750 Da / Num. of mol.: 1
Mutation: I129V,D322K,Y343L,R68S,M90V,Y227A,A282L,F327A,F338M,C358A,C116L,D200E
Source method: isolated from a genetically manipulated source
Details: Stabilized beta1-adrenergic receptor with AmpC beta-lactamase in ICL3,Stabilized beta1-adrenergic receptor with AmpC beta-lactamase in ICL3,Stabilized beta1-adrenergic receptor with AmpC beta-lactamase in ICL3
Source: (gene. exp.) Meleagris gallopavo (turkey), (gene. exp.) Escherichia coli K-12 (bacteria)
Gene: ADRB1, ampC, ampA, b4150, JW4111 / Production host: Homo sapiens (human)
References: UniProt: P07700, UniProt: P00811, beta-lactamase
#2: Chemical ChemComp-P32 / Cyanopindolol / 4-{[(2S)-3-(tert-butylamino)-2-hydroxypropyl]oxy}-3H-indole-2-carbonitrile


Mass: 287.357 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H21N3O2 / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: Fusion protein of stabilized beta1-adrenergic receptor containing Amp-C beta-lactamase in intracellular loop in complex with cyanopindolol
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Meleagris gallopavo (turkey)9103
31Escherichia coli (strain K12) (bacteria)83333
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
20.03 %DDM1
3100 mMNaCl1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 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 defocus max: 3000 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2729813
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37653 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 4.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025109
ELECTRON MICROSCOPYf_angle_d0.5326979
ELECTRON MICROSCOPYf_dihedral_angle_d5.914683
ELECTRON MICROSCOPYf_chiral_restr0.041792
ELECTRON MICROSCOPYf_plane_restr0.004867

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