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- EMDB-54355: Cryo-EM structure of a single-chain beta1-adrenoceptor - AmpC bet... -

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Basic information

Entry
Database: EMDB / ID: EMD-54355
TitleCryo-EM structure of a single-chain beta1-adrenoceptor - AmpC beta-lactamase fusion protein
Map data
Sample
  • Complex: Fusion protein of stabilized beta1-adrenergic receptor containing Amp-C beta-lactamase in intracellular loop in complex with cyanopindolol
    • Protein or peptide: Beta-1 adrenergic receptor,Beta-lactamase
  • Ligand: Cyanopindolol
KeywordsG protein-coupled receptor / cyanopindolol / AmpC beta-lactamase / fusion protein / cryo-EM / SIGNALING PROTEIN
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
Beta-lactamase / Beta-1 adrenergic receptor
Similarity search - Component
Biological speciesMeleagris gallopavo (turkey)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBenoit RM / Afanasyev P
Funding support Switzerland, 3 items
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, 2025-
Header (metadata) releaseJul 22, 2026-
Map releaseJul 22, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54355.png

Downloads & links

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Map

FileDownload / File: emd_54355.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 180 pix.
= 236.16 Å		1.31 Å/pix.
x 180 pix.
= 236.16 Å		1.31 Å/pix.
x 180 pix.
= 236.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.312 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.596874 - 0.90497595
Average (Standard dev.)0.0012883719 (±0.024584878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 236.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_54355_additional_1.map
Projections & Slices
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Half map: #2

Fileemd_54355_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_54355_half_map_2.map
Projections & Slices
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Sample components

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Entire : Fusion protein of stabilized beta1-adrenergic receptor containing...

EntireName: Fusion protein of stabilized beta1-adrenergic receptor containing Amp-C beta-lactamase in intracellular loop in complex with cyanopindolol
Components
  • Complex: Fusion protein of stabilized beta1-adrenergic receptor containing Amp-C beta-lactamase in intracellular loop in complex with cyanopindolol
    • Protein or peptide: Beta-1 adrenergic receptor,Beta-lactamase
  • Ligand: Cyanopindolol

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Supramolecule #1: Fusion protein of stabilized beta1-adrenergic receptor containing...

SupramoleculeName: Fusion protein of stabilized beta1-adrenergic receptor containing Amp-C beta-lactamase in intracellular loop in complex with cyanopindolol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Meleagris gallopavo (turkey)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Beta-1 adrenergic receptor,Beta-lactamase

MacromoleculeName: Beta-1 adrenergic receptor,Beta-lactamase / type: protein_or_peptide / ID: 1
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
Number of copies: 1 / Enantiomer: LEVO / EC number: beta-lactamase
Source (natural)Organism: Meleagris gallopavo (turkey)
Molecular weightTheoretical: 72.98375 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGAELLSQQW EAGMSLLMAL VVLLIVAGNV LVIAAIGSTQ RLQTLTNLFI TSLACADLVV GLLVVPFGAT LVVRGTWLWG SFLCELWTS LDVLCVTASV ETLCVIAIDR YLAITSPFRY QSLMTRARAK VIICTVWAIS ALVSFLPIMM HWWRDEDPQA L KCYQDPGC ...String:
MGAELLSQQW EAGMSLLMAL VVLLIVAGNV LVIAAIGSTQ RLQTLTNLFI TSLACADLVV GLLVVPFGAT LVVRGTWLWG SFLCELWTS LDVLCVTASV ETLCVIAIDR YLAITSPFRY QSLMTRARAK VIICTVWAIS ALVSFLPIMM HWWRDEDPQA L KCYQDPGC CEFVTNRAYA IASSIISFYI PLLIMIFVAL RVYREAKEQI NDIVHRTITP LIEQQKIPGM AVAVIYQGKP YY FTWGYAD IAKKQPVTQQ TLFELGSVSK TFTGVLGGDA IARGEIKLSD PTTKYWPELT AKQWNGITLL HLATYTAGGL PLQ VPDEVK SSSDLLRFYQ NWQPAWAPGT QRLYANSSIG LFGALAVKPS GLSFEQAMQT RVFQPLKLNH TWINVPPAEE KNYA WGYRE GKAVHVSPGA LDAEAYGVKS TIEDMARWVQ SNLKPLDINE KTLQQGIQLA QSRYWQTGDM YQGLGWEMLD WPVNP DSII NGSDNKIALA ARPVKAITPP TPAVRASWVH KTGATGGFGS YVAFIPEKEL GIVMLANKNY PNPARVDAAW QILNAL REH KALKTLGIIM GVFTLCWLPF FLVNIVNVFN RDLVPKWLFV AFNWLGYANS AMNPIILCRS PDFRKAFKRL LAFPRKA DR RLHGSGLEVL FQ

UniProtKB: Beta-1 adrenergic receptor, Beta-lactamase, Beta-1 adrenergic receptor

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Macromolecule #2: Cyanopindolol

MacromoleculeName: Cyanopindolol / type: ligand / ID: 2 / Number of copies: 1 / Formula: P32
Molecular weightTheoretical: 287.357 Da

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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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
50.0 mMHEPES
0.03 %DDM
100.0 mMNaCl
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2729813
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 37653
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-9rx1:
Cryo-EM structure of a single-chain beta1-adrenoceptor - AmpC beta-lactamase fusion protein

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