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- PDB-9t9p: Adenosine receptor A2a (A2AR)-beta-lactamase fusion bound to beta... -

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Basic information

Entry
Database: PDB / ID: 9t9p
TitleAdenosine receptor A2a (A2AR)-beta-lactamase fusion bound to beta-lactamase inhibitory protein II (BLIPII) and ZM241385
Components
  • Adenosine receptor A2a,Small exopenicillinase,Green fluorescent protein
  • Beta-lactamase inhibitory protein II
KeywordsMEMBRANE PROTEIN / G-protein coupled receptor / GPCR / fusion tag
Function / homology
Function and homology information


regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism ...regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / response to purine-containing compound / G protein-coupled adenosine receptor signaling pathway / NGF-independant TRKA activation / Surfactant metabolism / synaptic transmission, dopaminergic / type 5 metabotropic glutamate receptor binding / negative regulation of vascular permeability / synaptic transmission, cholinergic / intermediate filament / presynaptic active zone / positive regulation of urine volume / response to caffeine / blood circulation / sensory perception / positive regulation of glutamate secretion / eating behavior / inhibitory postsynaptic potential / regulation of calcium ion transport / alpha-actinin binding / beta-lactam antibiotic catabolic process / asymmetric synapse / axolemma / membrane depolarization / cellular defense response / prepulse inhibition / phagocytosis / neuron projection morphogenesis / positive regulation of synaptic transmission, glutamatergic / astrocyte activation / presynaptic modulation of chemical synaptic transmission / bioluminescence / positive regulation of long-term synaptic potentiation / positive regulation of synaptic transmission, GABAergic / central nervous system development / positive regulation of protein secretion / regulation of mitochondrial membrane potential / response to amphetamine / positive regulation of apoptotic signaling pathway / generation of precursor metabolites and energy / apoptotic signaling pathway / synaptic transmission, glutamatergic / excitatory postsynaptic potential / locomotory behavior / beta-lactamase activity / beta-lactamase / negative regulation of inflammatory response / vasodilation / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / blood coagulation / ubiquitin protein ligase activity / cell-cell signaling / adenylate cyclase-activating G protein-coupled receptor signaling pathway / presynaptic membrane / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / negative regulation of neuron apoptotic process / calmodulin binding / positive regulation of ERK1 and ERK2 cascade / postsynaptic membrane / response to xenobiotic stimulus / inflammatory response / negative regulation of cell population proliferation / response to antibiotic / neuronal cell body / apoptotic process / regulation of DNA-templated transcription / lipid binding / dendrite / protein-containing complex binding / glutamatergic synapse / enzyme binding / membrane / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
: / Regulator of chromosome condensation (RCC1) repeat / : / Regulator of chromosome condensation, RCC1 / Regulator of chromosome condensation (RCC1) repeat profile. / Regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II / Adenosine A2A receptor / Adenosine receptor / Beta-lactamase, class-A active site / Beta-lactamase class-A active site. ...: / Regulator of chromosome condensation (RCC1) repeat / : / Regulator of chromosome condensation, RCC1 / Regulator of chromosome condensation (RCC1) repeat profile. / Regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II / Adenosine A2A receptor / Adenosine receptor / Beta-lactamase, class-A active site / Beta-lactamase class-A active site. / Beta-lactamase class A, catalytic domain / Beta-lactamase enzyme family / Beta-lactamase, class-A / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Serpentine type 7TM GPCR chemoreceptor Srsx / Beta-lactamase/transpeptidase-like / Prokaryotic membrane lipoprotein lipid attachment site profile. / 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-ZMA / Beta-lactamase inhibitory protein II / Beta-lactamase / Adenosine receptor A2a / Green fluorescent protein
Similarity search - Component
Biological speciesStreptomyces exfoliatus (bacteria)
Homo sapiens (human)
Bacillus licheniformis (bacteria)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsShah, N.R. / Bisson, C. / Hutchin, A. / McFarlane, C.R. / Oosterlaken, M. / Pavic, A. / Zebisch, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2026
Title: A novel fusion tool to enable G protein-coupled receptor structure determination.
Authors: Nita R Shah / Mathieu Oosterlaken / Claudine Bisson / Mattia Bertinelli / Alicia M Churchill-Angus / Andrew Hutchin / Jola Kopec / Vadim Kotov / Ciaran R McFarlane / Erika Griss Pascualli / ...Authors: Nita R Shah / Mathieu Oosterlaken / Claudine Bisson / Mattia Bertinelli / Alicia M Churchill-Angus / Andrew Hutchin / Jola Kopec / Vadim Kotov / Ciaran R McFarlane / Erika Griss Pascualli / Ana Pavic / Matthias Zebisch / Cédric Fiez-Vandal / Edoardo Fabini / Stéphanie Duclos /
Abstract: Structure determination of G protein-coupled receptors (GPCRs) plays an important role in accelerating drug development against this medically important protein family. This study outlines the ...Structure determination of G protein-coupled receptors (GPCRs) plays an important role in accelerating drug development against this medically important protein family. This study outlines the development of a new fusion tool to enable structure determination of GPCRs in inactive conformations by cryo-EM. Initially, a PDB mining approach was applied to select eight naturally occurring proteins with the intention of fusing them into the intracellular loop 3 (ICL3) of GPCRs to create a suitable fiducial marker for cryo-EM workflows. During the selection process, candidates with known high-affinity protein binders were prioritized to enable a further increase in the protein mass of the fiducial marker. Fusion constructs were generated with adenosine receptor A (AR) and were assessed for expression and aggregation levels. For the two best-performing new fusion constructs, ligand binding was characterized to ensure that the fusion tag did not significantly affect protein behaviour. AR with a β-lactamase fusion in ICL3 and binding partner β-lactamase inhibitory protein II (BLIPII) was then selected to solve an antagonist-bound structure. The overall map was resolved to an average of 3.2 Å resolution with continuous helices connecting the β-lactamase to helices 5 and 6 of AR. Focused refinement of the AR region improved the local resolution and map detail in the orthosteric site, thereby allowing confident modelling of the antagonist ligand, ZM241385, which matches previously described X-ray crystallographic structures. This new fusion provides an alternative option for GPCR structure determination, with several potential benefits compared with existing tools, such as a more favourable position relative to the GPCR to reduce potential clashes.
History
DepositionNov 24, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Additional map / Part number: 3 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-lactamase inhibitory protein II
B: Adenosine receptor A2a,Small exopenicillinase,Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,3653
Polymers124,0282
Non-polymers3371
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-lactamase inhibitory protein II


Mass: 29044.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces exfoliatus (bacteria) / Gene: bliB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O87916
#2: Protein Adenosine receptor A2a,Small exopenicillinase,Green fluorescent protein


Mass: 94983.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Bacillus licheniformis (bacteria), (gene. exp.) Aequorea victoria (jellyfish)
Gene: ADORA2A, ADORA2, penP, blaP, GFP / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P29274, UniProt: P00808, UniProt: P42212
#3: Chemical ChemComp-ZMA / 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol


Mass: 337.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H15N7O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: antagonist*YM
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
IDNameTypeDetails (eV)Entity IDParent-IDSource
1A2AR-beta-lactamase fusion, BLIPII, ZM241385COMPLEXComplex of BLIPII bound to the beta-lactamase region of the chimeric A2AR-beta-lactamase fusion protein#1-#20MULTIPLE SOURCES
2A2AR-beta-lactamase fusionCOMPLEXbeta-lactamase fused into intracellular loop 3 of A2AR#11RECOMBINANT
3beta-lactamase inhibitory protein II (BLIPII)COMPLEX#21RECOMBINANT
4adenosine receptor A2a (A2AR)COMPLEX#12RECOMBINANT
5beta-lactamaseCOMPLEX#12RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
51NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21other entries (others)2787854
32other entries (others)2787854
43Streptomyces exfoliatus (bacteria)1905
44Homo sapiens (human)9606
55Bacillus licheniformis (bacteria)1402
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21other entries (others)2787854
32Spodoptera frugiperda (fall armyworm)7108Sf21
43Escherichia coli (E. coli)562BL21 (DE3)
44other sequences (unknown)28384
55other sequences (unknown)28384
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
140 mMHEPES1
2250 mMNaCl1
30.01 %LMNG1
40.001 %CHS1
SpecimenConc.: 0.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Complex was formed and isolated by size exclusion chromatography
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: 95 % / Chamber temperature: 277.15 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 magnification: 105000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.1 sec. / Electron dose: 49.71 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
2PHENIX"2.0_5885"model refinement
5cryoSPARC4.7.1CTF correction
10cryoSPARC4.7.1initial Euler assignment
11cryoSPARC4.7.1final Euler assignment
12cryoSPARC4.7.1classification
13cryoSPARC4.7.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 597000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16PS7

6ps7

16PS71PDBexperimental model
21JTD

1jtd

11JTD2PDBexperimental model
31AlphaFoldin silico model
RefinementHighest resolution: 3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026247
ELECTRON MICROSCOPYf_angle_d0.4588510
ELECTRON MICROSCOPYf_dihedral_angle_d14.3652209
ELECTRON MICROSCOPYf_chiral_restr0.04984
ELECTRON MICROSCOPYf_plane_restr0.0071081

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