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- EMDB-47040: Structure of rat beta-arrestin 1 by fiducial-assisted cryo-EM -

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Basic information

Entry
Database: EMDB / ID: EMD-47040
TitleStructure of rat beta-arrestin 1 by fiducial-assisted cryo-EM
Map dataStructure of rat beta-arrestin 1 by fiducial-assisted cryo-EM
Sample
  • Complex: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab LIGHT chain
    • Protein or peptide: Beta-arrestin-1,Soluble cytochrome b562
KeywordsGPCR signaling / arrestin / SIGNALING PROTEIN / SIGNALING PROTEIN-Immune System complex
Function / homology
Function and homology information


V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / regulation of inositol trisphosphate biosynthetic process / alpha-1B adrenergic receptor binding / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding ...V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / regulation of inositol trisphosphate biosynthetic process / alpha-1B adrenergic receptor binding / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding / Lysosome Vesicle Biogenesis / angiotensin receptor binding / AP-2 adaptor complex binding / Ub-specific processing proteases / MAP2K and MAPK activation / Golgi Associated Vesicle Biogenesis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / negative regulation of interleukin-8 production / regulation of G protein-coupled receptor signaling pathway / G protein-coupled receptor internalization / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / arrestin family protein binding / Thrombin signalling through proteinase activated receptors (PARs) / response to morphine / mitogen-activated protein kinase kinase binding / clathrin binding / stress fiber assembly / positive regulation of Rho protein signal transduction / pseudopodium / negative regulation of interleukin-6 production / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / phototransduction / positive regulation of insulin secretion involved in cellular response to glucose stimulus / insulin-like growth factor receptor binding / clathrin-coated pit / positive regulation of protein ubiquitination / negative regulation of protein ubiquitination / GTPase activator activity / nuclear estrogen receptor binding / phosphoprotein binding / G protein-coupled receptor binding / electron transport chain / negative regulation of ERK1 and ERK2 cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / positive regulation of protein phosphorylation / endocytosis / protein transport / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / regulation of apoptotic process / basolateral plasma membrane / molecular adaptor activity / dendritic spine / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / transmembrane transporter binding / postsynaptic membrane / periplasmic space / transcription coactivator activity / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / protein ubiquitination / endosome / positive regulation of MAPK cascade / postsynaptic density / G protein-coupled receptor signaling pathway / iron ion binding / response to xenobiotic stimulus / signaling receptor binding / positive regulation of cell population proliferation / ubiquitin protein ligase binding / heme binding / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / regulation of DNA-templated transcription / chromatin / glutamatergic synapse / enzyme binding / positive regulation of transcription by RNA polymerase II / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal ...Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Immunoglobulin E-set
Similarity search - Domain/homology
Soluble cytochrome b562 / Beta-arrestin-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsPakharukova N / Kahsai AW / Masoudi A / Lefkowitz RJ
Funding support United States, France, European Union, 6 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL016037 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)T32HL007101 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL16037-45S1 United States
Human Frontier Science Program (HFSP)LT000174/2018 France
European Molecular Biology Organization (EMBO)ALTF 1071-2017European Union
CitationJournal: bioRxiv / Year: 2025
Title: Small-molecule modulation of β-arrestins.
Authors: Alem W Kahsai / Natalia Pakharukova / Henry Y Kwon / Kunal S Shah / Jason G Liang-Lin / Caroline T Del Real / Paul J Shim / Mason A Lee / Van A Ngo / Bowie N Shreiber / Samuel Liu / Allison ...Authors: Alem W Kahsai / Natalia Pakharukova / Henry Y Kwon / Kunal S Shah / Jason G Liang-Lin / Caroline T Del Real / Paul J Shim / Mason A Lee / Van A Ngo / Bowie N Shreiber / Samuel Liu / Allison M Schwalb / Emmanuel F Espinoza / Brittany N Thomas / Cal A Kunzle / Jeffrey S Smith / Jialu Wang / Jihee Kim / Xingdong Zhang / Howard A Rockman / Alex R B Thomsen / Lindsay A M Rein / Lei Shi / Seungkirl Ahn / Ali Masoudi / Robert J Lefkowitz
Abstract: β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR ...β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study β-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt β-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and β-arrestin-dependent functions while sparing G protein-receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of β-arrestin1-a critical receptor-binding interface-stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for β-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.
History
DepositionSep 17, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47040.png

Downloads & links

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Map

FileDownload / File: emd_47040.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of rat beta-arrestin 1 by fiducial-assisted cryo-EM
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 300 pix.
= 414.72 Å		1.38 Å/pix.
x 300 pix.
= 414.72 Å		1.38 Å/pix.
x 300 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3824 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.02065325 - 2.038648
Average (Standard dev.)0.00024987196 (±0.009436742)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 414.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_47040_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_47040_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Beta-arrestin 1 with insertion of soluble cytochrome b562 bound t...

EntireName: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
Components
  • Complex: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab LIGHT chain
    • Protein or peptide: Beta-arrestin-1,Soluble cytochrome b562

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Supramolecule #1: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound t...

SupramoleculeName: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.904656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPK

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Macromolecule #2: anti-BRIL Fab LIGHT chain

MacromoleculeName: anti-BRIL Fab LIGHT chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.20982 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNR

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Macromolecule #3: Beta-arrestin-1,Soluble cytochrome b562

MacromoleculeName: Beta-arrestin-1,Soluble cytochrome b562 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 55.72716 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: LGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI ...String:
LGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI RKVQYAPEAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HG FDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLPTAET TRQFLMSDKP LHLEASLDKE IYYHGEPISV NVH VTNNTN KTVKKIKISV RQYADICLFN TAQYKCPVAM EEADDTVAPS STFCKVYTLT PFLANNREKR GLALDGKLKH EDTN LASST LLREGANREI LGIIVSYKVK VKLVVSRGDY KDDDDKSDVA VELPFTLMHP KPKEEPPHRE VPESETPVDT NLIEL DTND DDIVFEDFAR

UniProtKB: Beta-arrestin-1, Soluble cytochrome b562, Beta-arrestin-1

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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

Concentration7.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1g4m

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 479224
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

1g4m

chain_id: A, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: L, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: H, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: A, source_name: PDB, initial_model_type: experimental modelsoluble cytochrome b562 insert
Output model

PDB-9dng:
Structure of rat beta-arrestin 1 by fiducial-assisted cryo-EM

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