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- EMDB-56450: Focused map on beta-lactamase + BLIPII region -

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Basic information

Entry
Database: EMDB / ID: EMD-56450
TitleFocused map on beta-lactamase + BLIPII region
Map data
Sample
  • Complex: A2AR-beta-lactamase fusion, BLIPII, ZM241385
    • Complex: A2AR-beta-lactamase fusion
      • Complex: adenosine receptor A2a (A2AR)
      • Complex: beta-lactamase
    • Complex: beta-lactamase inhibitory protein II (BLIPII)
KeywordsG-protein coupled receptor / GPCR / fusion tag / MEMBRANE PROTEIN
Biological speciesother entries (others) / Streptomyces exfoliatus (bacteria) / Homo sapiens (human) / Bacillus licheniformis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsShah NR / Bisson C / Hutchin A / McFarlane CR / Oosterlaken M / Pavic A / Zebisch M
Funding support1 items
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
DepositionJan 21, 2026-
Header (metadata) releaseMay 27, 2026-
Map releaseMay 27, 2026-
UpdateMay 27, 2026-
Current statusMay 27, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56450.png

Downloads & links

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Map

FileDownload / File: emd_56450.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 300.6 Å		0.84 Å/pix.
x 360 pix.
= 300.6 Å		0.84 Å/pix.
x 360 pix.
= 300.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.396
Minimum - Maximum-2.0894775 - 4.0772943
Average (Standard dev.)0.00031070533 (±0.029294385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 300.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_56450_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_56450_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : A2AR-beta-lactamase fusion, BLIPII, ZM241385

EntireName: A2AR-beta-lactamase fusion, BLIPII, ZM241385
Components
  • Complex: A2AR-beta-lactamase fusion, BLIPII, ZM241385
    • Complex: A2AR-beta-lactamase fusion
      • Complex: adenosine receptor A2a (A2AR)
      • Complex: beta-lactamase
    • Complex: beta-lactamase inhibitory protein II (BLIPII)

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Supramolecule #1: A2AR-beta-lactamase fusion, BLIPII, ZM241385

SupramoleculeName: A2AR-beta-lactamase fusion, BLIPII, ZM241385 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Complex of BLIPII bound to the beta-lactamase region of the chimeric A2AR-beta-lactamase fusion protein
Source (natural)Organism: other entries (others)

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Supramolecule #2: A2AR-beta-lactamase fusion

SupramoleculeName: A2AR-beta-lactamase fusion / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: beta-lactamase fused into intracellular loop 3 of A2AR
Source (natural)Organism: other entries (others)

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Supramolecule #3: beta-lactamase inhibitory protein II (BLIPII)

SupramoleculeName: beta-lactamase inhibitory protein II (BLIPII) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Streptomyces exfoliatus (bacteria)

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Supramolecule #4: adenosine receptor A2a (A2AR)

SupramoleculeName: adenosine receptor A2a (A2AR) / type: complex / ID: 4 / Parent: 2 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: beta-lactamase

SupramoleculeName: beta-lactamase / type: complex / ID: 5 / Parent: 2 / Macromolecule list: #1
Source (natural)Organism: Bacillus licheniformis (bacteria)

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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

Concentration0.38 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
40.0 mMHEPES
250.0 mMNaCl
0.01 %LMNG
0.001 %CHS
GridModel: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsComplex was formed and isolated by size exclusion chromatography

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.1 sec. / Average electron dose: 49.71 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 597000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial model
PDB IDChain

6ps7

source_name: PDB, initial_model_type: experimental model

1jtd

source_name: PDB, initial_model_type: experimental model
source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient

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