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- PDB-8xgr: ETB-eGt complex bound to endothelin-1 -

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Basic information

Entry
Database: PDB / ID: 8xgr
TitleETB-eGt complex bound to endothelin-1
Components
  • Camelid antibody VHH fragment
  • Endothelin receptor type B
  • Endothelin-1Endothelin 1
  • Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,eGt-alpha
  • Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
KeywordsPEPTIDE BINDING PROTEIN/IMMUNE SYSTEM / SIGNALING PROTEIN / PEPTIDE BINDING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of prostaglandin-endoperoxide synthase activity / endothelin A receptor binding / protein kinase C deactivation / cellular response to human chorionic gonadotropin stimulus / meiotic cell cycle process involved in oocyte maturation / phospholipase D-activating G protein-coupled receptor signaling pathway / endothelin B receptor binding / rhythmic excitation / peptide hormone secretion / neural crest cell fate commitment ...positive regulation of prostaglandin-endoperoxide synthase activity / endothelin A receptor binding / protein kinase C deactivation / cellular response to human chorionic gonadotropin stimulus / meiotic cell cycle process involved in oocyte maturation / phospholipase D-activating G protein-coupled receptor signaling pathway / endothelin B receptor binding / rhythmic excitation / peptide hormone secretion / neural crest cell fate commitment / sympathetic neuron axon guidance / body fluid secretion / glomerular endothelium development / vein smooth muscle contraction / noradrenergic neuron differentiation / response to prostaglandin F / positive regulation of renal sodium excretion / leukocyte activation / positive regulation of sarcomere organization / histamine secretion / rough endoplasmic reticulum lumen / positive regulation of chemokine-mediated signaling pathway / positive regulation of odontogenesis / maternal process involved in parturition / regulation of glucose transmembrane transport / pharyngeal arch artery morphogenesis / endothelin receptor signaling pathway involved in heart process / semaphorin-plexin signaling pathway involved in axon guidance / epithelial fluid transport / podocyte differentiation / cardiac neural crest cell migration involved in outflow tract morphogenesis / negative regulation of hormone secretion / response to leptin / endothelin receptor signaling pathway / response to ozone / Weibel-Palade body / renal sodium ion absorption / positive regulation of cell growth involved in cardiac muscle cell development / artery smooth muscle contraction / glomerular filtration / axonogenesis involved in innervation / positive regulation of cation channel activity / positive regulation of prostaglandin secretion / cellular response to follicle-stimulating hormone stimulus / cellular response to luteinizing hormone stimulus / regulation of pH / negative regulation of nitric-oxide synthase biosynthetic process / cellular response to mineralocorticoid stimulus / positive regulation of urine volume / negative regulation of blood coagulation / respiratory gaseous exchange by respiratory system / basal part of cell / positive regulation of smooth muscle contraction / positive regulation of hormone secretion / regulation of systemic arterial blood pressure by endothelin / vasoconstriction / protein kinase C-activating G protein-coupled receptor signaling pathway / embryonic heart tube development / superoxide anion generation / axon extension / dorsal/ventral pattern formation / negative regulation of protein metabolic process / positive regulation of neutrophil chemotaxis / middle ear morphogenesis / positive regulation of signaling receptor activity / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / cellular response to glucocorticoid stimulus / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (12/13) signalling events / G beta:gamma signalling through BTK / cartilage development / ADP signalling through P2Y purinoceptor 12 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / G alpha (z) signalling events / Extra-nuclear estrogen signaling / G alpha (s) signalling events / prostaglandin biosynthetic process / G alpha (q) signalling events / nitric oxide transport / cellular response to fatty acid / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / positive regulation of heart rate / cellular response to organic substance / branching involved in blood vessel morphogenesis / Vasopressin regulates renal water homeostasis via Aquaporins / response to testosterone / response to dexamethasone
Similarity search - Function
Endothelin-like toxin / Endothelin-like toxin, conserved site / Endothelin / Endothelin family / Endothelin family signature. / Endothelin / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily ...Endothelin-like toxin / Endothelin-like toxin, conserved site / Endothelin / Endothelin family / Endothelin family signature. / Endothelin / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs
Similarity search - Domain/homology
Endothelin-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesBos taurus (cattle)
Rattus rattus (black rat)
Lama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsOshima, H.S. / Sano, F.K. / Akasaka, H. / Iwama, A. / Shihoya, W. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H05037 Japan
CitationJournal: Biochem Biophys Res Commun / Year: 2024
Title: Optimizing cryo-EM structural analysis of G-coupling receptors via engineered G and Nb35 application.
Authors: Hidetaka S Oshima / Fumiya K Sano / Hiroaki Akasaka / Aika Iwama / Wataru Shihoya / Osamu Nureki /
Abstract: Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in ...Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-G complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-G complexes remain both challenging and resource-intensive. By employing eGα, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGα complex was previously reported. Using this modified G protein, we determined the structure of the ET-eG complex bound to the modified Nb35. The determined structure of ET receptor was the same as the previously reported ET-G complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-G complexes.
History
DepositionDec 15, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,eGt-alpha
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
N: Camelid antibody VHH fragment
R: Endothelin receptor type B
L: Endothelin-1


Theoretical massNumber of molelcules
Total (without water)176,8555
Polymers176,8555
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,eGt-alpha / G gamma-I


Mass: 48998.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63212
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Mass: 40470.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus rattus (black rat) / Production host: Spodoptera frugiperda (fall armyworm)
#3: Antibody Camelid antibody VHH fragment


Mass: 17395.631 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Spodoptera frugiperda (fall armyworm)
#4: Protein Endothelin receptor type B


Mass: 67492.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EDNRB, ETRB / Production host: Spodoptera frugiperda (fall armyworm)
#5: Protein/peptide Endothelin-1 / Endothelin 1 / Preproendothelin-1 / PPET1


Mass: 2497.951 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P05305

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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: Complex of Endothelin-1, ETB, eGt trimer and Nb35* / Type: COMPLEX / Entity ID: #4-#5, #2-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52285 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038456
ELECTRON MICROSCOPYf_angle_d0.5711437
ELECTRON MICROSCOPYf_dihedral_angle_d14.0813076
ELECTRON MICROSCOPYf_chiral_restr0.0391301
ELECTRON MICROSCOPYf_plane_restr0.0051443

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