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- PDB-21ty: Cryo-EM structure of Free fatty acid receptor 2 (FFA2)-ARK1 with ... -

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Basic information

Entry
Database: PDB / ID: 21ty
TitleCryo-EM structure of Free fatty acid receptor 2 (FFA2)-ARK1 with GLPG0974
ComponentsFree fatty acid receptor 2,human free fatty acid receptor 2 (FFA2) fused with de novo designed ARK1
KeywordsMEMBRANE PROTEIN / GPCR / FFA2 / Free fatty acid receptor 2 / GLPG0974 / ARK1
Function / homology
Function and homology information


positive regulation of acute inflammatory response to non-antigenic stimulus / regulation of peptide hormone secretion / regulation of acute inflammatory response / leukocyte chemotaxis involved in inflammatory response / mucosal immune response / Free fatty acid receptors / lipid storage / cell surface pattern recognition receptor signaling pathway / positive regulation of cytokine production involved in immune response / cellular response to fatty acid ...positive regulation of acute inflammatory response to non-antigenic stimulus / regulation of peptide hormone secretion / regulation of acute inflammatory response / leukocyte chemotaxis involved in inflammatory response / mucosal immune response / Free fatty acid receptors / lipid storage / cell surface pattern recognition receptor signaling pathway / positive regulation of cytokine production involved in immune response / cellular response to fatty acid / fat cell differentiation / positive regulation of chemokine production / cell projection / positive regulation of interleukin-8 production / G protein-coupled receptor activity / phospholipase C-activating G protein-coupled receptor signaling pathway / glucose homeostasis / G alpha (q) signalling events / G protein-coupled receptor signaling pathway / lipid binding / plasma membrane
Similarity search - Function
G protein-coupled receptor 40-related receptor / 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
: / Free fatty acid receptor 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.93 Å
AuthorsKojima, A. / Kawakami, K. / Narita, T. / Kugawa, M. / Hayashi, K. / Fukuda, M. / Kato, H.E.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJAX222F Japan
Japan Science and TechnologyJPMJPR24OF Japan
Japan Society for the Promotion of Science (JSPS)25K09525 Japan
Japan Society for the Promotion of Science (JSPS)24H02262 Japan
Japan Society for the Promotion of Science (JSPS)25H01338 Japan
Japan Society for the Promotion of Science (JSPS)JP24KJ0981 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Universal pipeline for high-resolution GPCR structure determination.
Authors: Asato Kojima / Kouki Kawakami / Naoya Kobayashi / Kazuhiro Kobayashi / Toshiki E Matsui / Kohei Uemoto / Yuzhong Gu / Tomohiro J Narita / Mai Kugawa / Masahiro Fukuda / Hideaki E Kato /
Abstract: G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain ...G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain difficult because current fusion-based strategies often require extensive experimental screening to identify rigid constructs suitable for high-resolution reconstruction. Here we introduce a universal pipeline that integrates an in silico fusion construct screening program, NOAH (nonexperimental, artificial-intelligence-assisted, high-throughput construct screening for structural analysis), with a de novo designed fusion protein, ARK1 (artificially designed fiducial marker). NOAH enabled structure determination of vasopressin V2 receptor bound to the antagonist tolvaptan or partial agonist OPC51803 and bradykinin B2 receptor bound to the antagonist icatibant, revealing receptor activation and inhibition mechanisms. Coupling NOAH to ARK1 improved the V2 receptor-tolvaptan map and enabled high-resolution structures of lysophosphatidic acid receptor 2 bound to Ki16425 and free fatty acid receptor 2 bound to GLPG0974. NOAH-ARK1 minimizes trial-and-error construct optimization and provides a broadly applicable route for GPCR structural analysis and drug discovery.
History
DepositionDec 26, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 9, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 9, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Free fatty acid receptor 2,human free fatty acid receptor 2 (FFA2) fused with de novo designed ARK1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,0502
Polymers85,5651
Non-polymers4851
Water1267
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 Free fatty acid receptor 2,human free fatty acid receptor 2 (FFA2) fused with de novo designed ARK1 / G-protein coupled receptor 43


Mass: 85565.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FFAR2, FFA2, GPCR43, GPR43 / Production host: Homo sapiens (human) / References: UniProt: O15552
#2: Chemical ChemComp-A1LYD / GLPG-0974 / 4-[[(2~{R})-1-(1-benzothiophen-3-ylcarbonyl)-2-methyl-azetidin-2-yl]carbonyl-[(3-chlorophenyl)methyl]amino]butanoic acid


Mass: 484.995 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H25ClN2O4S / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O
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

ComponentName: Cryo-EM structure of Free fatty acid receptor 2 (FFA2)-ARK1 with GLPG0974
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 49.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
12cryoSPARC3D reconstruction
19REFMAC5.8.0425model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196932 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 2.93→2.93 Å / Cor.coef. Fo:Fc: 0.811 / WRfactor Rwork: 0.421 / SU B: 16.969 / SU ML: 0.273 / Average fsc free: 0 / Average fsc overall: 0.7204 / Average fsc work: 0.7204 / ESU R: 0.453
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.4208 63424 -
all0.421 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 112.306 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0125467
ELECTRON MICROSCOPYr_bond_other_d00.0165547
ELECTRON MICROSCOPYr_ext_dist_refined_b0.0020.189051
ELECTRON MICROSCOPYr_angle_refined_deg1.5991.8787387
ELECTRON MICROSCOPYr_angle_other_deg0.6441.76112752
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.0565660
ELECTRON MICROSCOPYr_dihedral_angle_2_deg11.2663.360
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.535101062
ELECTRON MICROSCOPYr_dihedral_angle_6_deg10.5310236
ELECTRON MICROSCOPYr_chiral_restr0.0860.2873
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.026297
ELECTRON MICROSCOPYr_gen_planes_other0.0030.021205
ELECTRON MICROSCOPYr_nbd_refined0.2140.21294
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1480.25045
ELECTRON MICROSCOPYr_nbtor_refined0.1650.22749
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0610.23111
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1340.2116
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0290.21
ELECTRON MICROSCOPYr_mcbond_it10.00110.0742646
ELECTRON MICROSCOPYr_mcbond_other10.00110.0742646
ELECTRON MICROSCOPYr_mcangle_it15.77718.1623304
ELECTRON MICROSCOPYr_mcangle_other15.77518.1663305
ELECTRON MICROSCOPYr_scbond_it13.12612.1492821
ELECTRON MICROSCOPYr_scbond_other13.12412.1472822
ELECTRON MICROSCOPYr_scangle_it22.22321.3764081
ELECTRON MICROSCOPYr_scangle_other22.2221.3734082
ELECTRON MICROSCOPYr_lrange_it35.542221.43389853
ELECTRON MICROSCOPYr_lrange_other35.542221.43189854
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
3-3.0781.46746911.46746910.2961.467
3.078-3.1620.95945250.95945250.3590.959
3.162-3.2540.91845390.91845390.4460.918
3.254-3.3540.84142740.84142740.5240.841
3.354-3.4640.72841840.72841840.5780.728
3.464-3.5850.59340740.59340740.6860.593
3.585-3.7210.39438640.39438640.7930.394
3.721-3.8720.31837410.31837410.8620.318
3.872-4.0440.33636020.33636020.9040.336
4.044-4.2410.36535010.36535010.9370.365
4.241-4.4710.36632680.36632680.9530.366
4.471-4.7410.35331200.35331200.9550.353
4.741-5.0680.32228910.32228910.9520.322
5.068-5.4740.30327060.30327060.9260.303
5.474-5.9950.32225040.32225040.8590.322
5.995-6.7010.35722320.35722320.8250.357
6.701-7.7340.37319980.37319980.8230.373
7.734-9.4630.32616730.32616730.8880.326
9.463-13.3470.28313150.28313150.9270.283
13.347-127.820.8537220.8537220.9530.853

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