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- PDB-9xqb: Cryo-EM structure of the human A2A adenosine receptor in complex ... -

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Basic information

Entry
Database: PDB / ID: 9xqb
TitleCryo-EM structure of the human A2A adenosine receptor in complex with a Fab antibody fragment
Components
  • A2A receptor-BRIL
  • Fab Heavy chain
  • Fab Light chain
KeywordsMEMBRANE PROTEIN / Cryo-EM Lip-MS
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsMiyashita, Y. / Konno, R. / Ogasawara, S. / Okuda, Y. / Takamuku, Y. / Moriya, T. / Saito, T. / Murata, T. / Ohara, O. / Kawashima, Y.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23K27158, 25K18417 Japan
Japan Agency for Medical Research and Development (AMED)JP25ama121013 Japan
Kazusa DNA Research Institute Foundation Japan
CitationJournal: ACS Omega / Year: 2026
Title: Rapid and Label-Free Structural Proteomics Using One-Step Swift Trypsin LiP-MS.
Authors: Yasuomi Miyashita / Ryo Konno / Satoshi Ogasawara / Yusei Okuda / Yuuki Takamuku / Toshio Moriya / Tetsuichiro Saito / Takeshi Murata / Osamu Ohara / Yusuke Kawashima /
Abstract: Limited proteolysis mass spectrometry (LiP-MS) is a powerful approach for probing protein conformational changes on a proteome-wide scale. However, conventional workflows rely on a two-step digestion ...Limited proteolysis mass spectrometry (LiP-MS) is a powerful approach for probing protein conformational changes on a proteome-wide scale. However, conventional workflows rely on a two-step digestion with proteinase K and trypsin, which increases complexity and reduces reproducibility and sensitivity. This study aimed to develop a simplified one-step protocol, termed Swift Trypsin LiP-MS (STLiP-MS), which uses a trypsin-immobilized spin column and high-speed centrifugation to achieve rapid and reproducible surface-limited proteolysis. Using HEK293 cell extracts, STLiP-MS identified 286 proteins exhibiting conformational changes upon phosphatase inhibition, including 37 enriched in phosphatase-related Gene Ontology categories. The method improvements, including suppression of predigestion and immediate enzyme inactivation, further increased sensitivity, enabling the detection of 799 proteins with structural alterations, of which 77 were enriched in phosphatase-related categories. Comparison with the single-pot solid-phase-enhanced sample preparation (SP3) method confirmed that these changes originated from structure-selective proteolysis and were not detectable under fully denaturing conditions. To demonstrate its broader applicability, we applied STLiP-MS to the adenosine A receptor (A-BRIL) and observed antibody-induced protection of extracellular loop 2 (residues 147-176). Cryogenic electron microscopy validated Fab fragment binding to the same region, confirming the correspondence between STLiP-MS signals and actual antibody-antigen interfaces. Collectively, these results show that STLiP-MS is a rapid and robust platform that enables sensitive, label-free detection of local structural changes under near-physiological conditions and accurate prediction of protein-protein interaction sites. This method holds great promise for applications in structural proteomics and drug target identification.
History
DepositionNov 18, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 7, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 7, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Jan 7, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0Jan 7, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 4, 2026Group: Data collection / Database references / Category: citation / em_admin
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Revision 1.1Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: citation / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Fab Light chain
C: A2A receptor-BRIL
A: Fab Heavy chain


Theoretical massNumber of molelcules
Total (without water)77,1513
Polymers77,1513
Non-polymers00
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: Antibody Fab Light chain


Mass: 13761.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#2: Protein A2A receptor-BRIL


Mass: 47924.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody Fab Heavy chain


Mass: 15465.118 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
Has protein modificationN

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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: A2A receptor BRIL Fab complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172394 / Symmetry type: POINT
RefinementHighest resolution: 3.45 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0022976
ELECTRON MICROSCOPYf_angle_d0.4924067
ELECTRON MICROSCOPYf_dihedral_angle_d4.508475
ELECTRON MICROSCOPYf_chiral_restr0.04486
ELECTRON MICROSCOPYf_plane_restr0.003533

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