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- EMDB-67107: Cryo-EM structure of the human A2A adenosine receptor in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-67107
TitleCryo-EM structure of the human A2A adenosine receptor in complex with a Fab antibody fragment
Map data
Sample
  • Complex: A2A receptor BRIL Fab complex
    • Protein or peptide: Fab Light chain
    • Protein or peptide: A2A receptor-BRIL
    • Protein or peptide: Fab Heavy chain
KeywordsCryo-EM Lip-MS / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle 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, 3 items
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, 2025-
Header (metadata) releaseJan 7, 2026-
Map releaseJan 7, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_67107.png

Downloads & links

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Map

FileDownload / File: emd_67107.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 360 pix.
= 270. Å		0.75 Å/pix.
x 360 pix.
= 270. Å		0.75 Å/pix.
x 360 pix.
= 270. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.75 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.25627914 - 0.40623793
Average (Standard dev.)0.00006391761 (±0.00767817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_67107_msk_1.map
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Half map: #2

Fileemd_67107_half_map_1.map
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Half map: #1

Fileemd_67107_half_map_2.map
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Sample components

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Entire : A2A receptor BRIL Fab complex

EntireName: A2A receptor BRIL Fab complex
Components
  • Complex: A2A receptor BRIL Fab complex
    • Protein or peptide: Fab Light chain
    • Protein or peptide: A2A receptor-BRIL
    • Protein or peptide: Fab Heavy chain

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Supramolecule #1: A2A receptor BRIL Fab complex

SupramoleculeName: A2A receptor BRIL Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Fab Light chain

MacromoleculeName: Fab Light chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.761061 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DIQLTQSPAV LSVSPGERVS FSCRASQTIG TSIHWYQQRT NGSPRLLVKF ASESISGIPS RFSGSGSGTD FTLTINSVES EDIADYYCQ QSNSWPYTFG GGTKLEIKRA DAAPTVSKGE FQHTGGRY

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Macromolecule #2: A2A receptor-BRIL

MacromoleculeName: A2A receptor-BRIL / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.924848 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DYKDDDDKPI MGSSVYITVE LAIAVLAILG NVLVCWAVWL NSNLQNVTNY FVVSLAAADI AVGVLAIPFA ITISTGFCAA CHGCLFIAC FVLVLEQSRI FSLLAIAIDR YIAIRIPLRY NGLVTGTRAK GIIAICWVLS FAIGLTPMLG WNNCGQPKEG K QHSQGCGE ...String:
DYKDDDDKPI MGSSVYITVE LAIAVLAILG NVLVCWAVWL NSNLQNVTNY FVVSLAAADI AVGVLAIPFA ITISTGFCAA CHGCLFIAC FVLVLEQSRI FSLLAIAIDR YIAIRIPLRY NGLVTGTRAK GIIAICWVLS FAIGLTPMLG WNNCGQPKEG K QHSQGCGE GQVACLFEDV VPMNYMVYFN FFACVLVPLL LMLGVYLRIF LAARRQLADL EDNWETLNDN LKVIEKADNA AQ VKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HGFDILVGQI DDALKLANEG KVKEAQAAAE QLKTTRNAYI QKY LERARS TLQKEVHAAK SLAIIVGLFA LCWLPLHIIN CFTFFCPDCS HAPLWLMYLA IVLSHTNSVV NPFIYAYRIR EFRQ TFRKI IRSHVLRQQE PFKAGGSGSG LEVLFQ

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Macromolecule #3: Fab Heavy chain

MacromoleculeName: Fab Heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 15.465118 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
EVKLVESGPE LKKPGETVKI SCKASGYSFT NYGINWMKQA PGEGLEWMGW INTYTGEATY DDDFKGRFAF SLETSASTAY LQIINLKNE DMATYFCSRR GAYYRYDGYF YTMDFWGQGT SVTVSSKGEF QHTGGRY

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 172394
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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