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- EMDB-9375: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-bet... -

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Basic information

Entry
Database: EMDB / ID: EMD-9375
TitleStabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Map dataStabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Sample
  • Complex: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
    • Complex: Nanobody 32Single-domain antibody
      • Protein or peptide: Nanobody 32Single-domain antibody
    • Complex: Beta-arrestin-1Arrestin
      • Protein or peptide: Beta-arrestin-1Arrestin
    • Complex: Fab30
      • Protein or peptide: Fab30 Heavy Chain
      • Protein or peptide: Fab30 Light Chain
    • Complex: Vasopressin 2 Tail
      • Protein or peptide: Vasopressin V2 receptorVasopressin receptor 2
Function / homology
Function and homology information


renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / MAP2K and MAPK activation / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / Activation of SMO / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / AP-2 adaptor complex binding ...renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / MAP2K and MAPK activation / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / Activation of SMO / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / AP-2 adaptor complex binding / clathrin heavy chain binding / clathrin coat of coated pit / Ub-specific processing proteases / desensitization of G protein-coupled receptor signaling pathway / hemostasis / positive regulation of systemic arterial blood pressure / telencephalon development / Cargo recognition for clathrin-mediated endocytosis / inositol hexakisphosphate binding / Clathrin-mediated endocytosis / clathrin-dependent endocytosis / G protein-coupled receptor internalization / acetylcholine receptor binding / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (s) signalling events / positive regulation of intracellular signal transduction / clathrin binding / negative regulation of Notch signaling pathway / pseudopodium / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of receptor internalization / endocytic vesicle / small molecule binding / positive regulation of vasoconstriction / cellular response to hormone stimulus / activation of adenylate cyclase activity / visual perception / response to cytokine / G protein-coupled receptor binding / peptide binding / clathrin-coated endocytic vesicle membrane / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / receptor internalization / Vasopressin regulates renal water homeostasis via Aquaporins / protein transport / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / cytoplasmic vesicle / G alpha (s) signalling events / ubiquitin-dependent protein catabolic process / molecular adaptor activity / positive regulation of ERK1 and ERK2 cascade / endosome / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / positive regulation of cell population proliferation / positive regulation of gene expression / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / signal transduction / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Vasopressin V2 receptor / Vasopressin receptor / 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 ...Vasopressin V2 receptor / Vasopressin receptor / 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 / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Immunoglobulin E-set
Similarity search - Domain/homology
Beta-arrestin-1 / Vasopressin V2 receptor
Similarity search - Component
Biological speciesLama glama (llama) / Bos taurus (cattle) / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsNguyen AH / Thomsen ARB / Cahill TJ / des Georges A / Lefkowitz RJ
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)2R01-HL016037-47 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.
Authors: Anthony H Nguyen / Alex R B Thomsen / Thomas J Cahill / Rick Huang / Li-Yin Huang / Tara Marcink / Oliver B Clarke / Søren Heissel / Ali Masoudi / Danya Ben-Hail / Fadi Samaan / Venkata P ...Authors: Anthony H Nguyen / Alex R B Thomsen / Thomas J Cahill / Rick Huang / Li-Yin Huang / Tara Marcink / Oliver B Clarke / Søren Heissel / Ali Masoudi / Danya Ben-Hail / Fadi Samaan / Venkata P Dandey / Yong Zi Tan / Chuan Hong / Jacob P Mahoney / Sarah Triest / John Little / Xin Chen / Roger Sunahara / Jan Steyaert / Henrik Molina / Zhiheng Yu / Amedee des Georges / Robert J Lefkowitz /
Abstract: Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to ...Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.
History
DepositionDec 26, 2018-
Header (metadata) releaseSep 25, 2019-
Map releaseNov 20, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.22
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.22
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ni2
  • Surface level: 0.22
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9375.png

Downloads & links

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Map

FileDownload / File: emd_9375.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 240 pix.
= 249.6 Å		1.04 Å/pix.
x 240 pix.
= 249.6 Å		1.04 Å/pix.
x 240 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22 / Movie #1: 0.22
Minimum - Maximum-0.34983063 - 0.96552765
Average (Standard dev.)0.000315015 (±0.025749706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z249.600249.600249.600
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.3500.9660.000

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Supplemental data

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Sample components

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Entire : Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-bet...

EntireName: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
Components
  • Complex: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
    • Complex: Nanobody 32Single-domain antibody
      • Protein or peptide: Nanobody 32Single-domain antibody
    • Complex: Beta-arrestin-1Arrestin
      • Protein or peptide: Beta-arrestin-1Arrestin
    • Complex: Fab30
      • Protein or peptide: Fab30 Heavy Chain
      • Protein or peptide: Fab30 Light Chain
    • Complex: Vasopressin 2 Tail
      • Protein or peptide: Vasopressin V2 receptorVasopressin receptor 2

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Supramolecule #1: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-bet...

SupramoleculeName: Stabilized beta-arrestin 1-V2T subcomplex of a GPCR-G protein-beta-arrestin mega-complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Nanobody 32

SupramoleculeName: Nanobody 32 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Beta-arrestin-1

SupramoleculeName: Beta-arrestin-1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #4: Fab30

SupramoleculeName: Fab30 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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Supramolecule #5: Vasopressin 2 Tail

SupramoleculeName: Vasopressin 2 Tail / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Nanobody 32

MacromoleculeName: Nanobody 32 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.665136 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCVVSGFFFD TVTMAWYRRA PGKHRELVAS ATAGGTTTYA DSVKDRFTIS RDNAKNTVYL QMNSLKPED TAVYYCNTFV RSLSWGQGTQ VTVSSHHHHH HEPEA

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Macromolecule #2: Beta-arrestin-1

MacromoleculeName: Beta-arrestin-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 44.309578 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVEPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI ...String:
MGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVEPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI RKVQYAPERP GPQPTAETTR QFLMSDKPLH LEASLDKEIY YHGEPISVNV HVTNNTNKTV KKIKISVRQY AD ICLFNTA QYKCPVAMEE ADDTVAPSST FCKVYTLTPF LANNREKRGL ALDGKLKHED TNLASSTLLR EGANREILGI IVS YKVKVK LVVSRGGLLG DLASSDVAVE LPFTLMHPKP KEEPPHREVP EHETPVDTNL IELDTNDDDI VFEDFAR

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Macromolecule #3: Fab30 Heavy Chain

MacromoleculeName: Fab30 Heavy Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.512354 KDa
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYYGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSRQFWYSG LDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYYGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARSRQFWYSG LDYWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THHHHHHHH

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Macromolecule #4: Fab30 Light Chain

MacromoleculeName: Fab30 Light Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.435064 KDa
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYKYVPVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYKYVPVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #5: Vasopressin V2 receptor

MacromoleculeName: Vasopressin V2 receptor / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.115718 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
ARGRTPPSLG PQDE(SEP)C(TPO)(TPO)A(SEP) (SEP)(SEP)LAKD

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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
GridDetails: unidentified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 104.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 230021

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