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- PDB-8avd: Cryo-EM structure for a 3:3 complex between mouse leptin and the ... -

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Basic information

Entry
Database: PDB / ID: 8avd
TitleCryo-EM structure for a 3:3 complex between mouse leptin and the mouse LEP-R ectodomain (local refinement)
Components
  • Leptin
  • Leptin receptor
KeywordsCYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance
Function / homology
Function and homology information


negative regulation of metabolic process / negative regulation of locomotor rhythm / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of eating behavior / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / leptin receptor activity / negative regulation of appetite by leptin-mediated signaling pathway ...negative regulation of metabolic process / negative regulation of locomotor rhythm / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of eating behavior / regulation of lipoprotein lipid oxidation / cellular response to L-ascorbic acid / positive regulation of fat cell apoptotic process / negative regulation of glutamine transport / leptin receptor activity / negative regulation of appetite by leptin-mediated signaling pathway / negative regulation of glucagon secretion / regulation of endothelial cell proliferation / regulation of natural killer cell proliferation / leptin receptor binding / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / protein-hormone receptor activity / positive regulation of luteinizing hormone secretion / regulation of natural killer cell mediated cytotoxicity / bone growth / regulation of natural killer cell activation / positive regulation of monoatomic ion transport / glycerol biosynthetic process / elastin metabolic process / leptin-mediated signaling pathway / positive regulation of follicle-stimulating hormone secretion / regulation of steroid biosynthetic process / regulation of intestinal cholesterol absorption / regulation of bone remodeling / regulation of brown fat cell differentiation / positive regulation of peroxisome proliferator activated receptor signaling pathway / adult feeding behavior / positive regulation of hepatic stellate cell activation / response to leptin / regulation of nitric-oxide synthase activity / bone mineralization involved in bone maturation / sexual reproduction / regulation of feeding behavior / regulation of lipid biosynthetic process / activation of protein kinase C activity / negative regulation of cartilage development / fatty acid catabolic process / ovulation from ovarian follicle / negative regulation of appetite / positive regulation of developmental growth / leukocyte tethering or rolling / energy reserve metabolic process / regulation of metabolic process / prostaglandin secretion / negative regulation of glucose import / bile acid metabolic process / tyrosine phosphorylation of STAT protein / cellular response to leptin stimulus / hormone metabolic process / regulation of protein localization to nucleus / cardiac muscle hypertrophy / aorta development / intestinal absorption / insulin secretion / regulation of fat cell differentiation / positive regulation of p38MAPK cascade / cytokine receptor activity / peptide hormone receptor binding / negative regulation of vasoconstriction / eating behavior / regulation of gluconeogenesis / glycogen metabolic process / cytokine binding / fatty acid beta-oxidation / central nervous system neuron development / regulation of cytokine production involved in inflammatory response / positive regulation of insulin secretion involved in cellular response to glucose stimulus / peptide hormone binding / regulation of insulin secretion / response to dietary excess / negative regulation of lipid storage / T cell differentiation / positive regulation of TOR signaling / response to vitamin E / glial cell proliferation / regulation of angiogenesis / adipose tissue development / negative regulation of gluconeogenesis / phagocytosis / energy homeostasis / cellular response to retinoic acid / positive regulation of insulin receptor signaling pathway / positive regulation of T cell proliferation / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of interleukin-12 production / negative regulation of autophagy / cholesterol metabolic process / response to activity / positive regulation of interleukin-8 production / gluconeogenesis / female pregnancy / determination of adult lifespan / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein phosphorylation / response to insulin / placenta development
Similarity search - Function
Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / Obesity receptor immunoglobulin like domain / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Short hematopoietin receptor, family 1, conserved site / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core ...Leptin / Leptin / Leptin receptor, immunoglobulin-like domain / Obesity receptor immunoglobulin like domain / Immunoglobulin C2-set-like, ligand-binding / Ig-like C2-type domain / Short hematopoietin receptor, family 1, conserved site / Long hematopoietin receptor, Gp130 family 2, conserved site / Long hematopoietin receptor, gp130 family signature. / Four-helical cytokine-like, core / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like fold
Similarity search - Domain/homology
NICKEL (II) ION / Leptin / Leptin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.42 Å
AuthorsVerstraete, K. / Savvides, S.N. / Verschueren, K.G. / Tsirigotaki, A.
Funding support Belgium, 1items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0G0619N Belgium
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanism of receptor assembly via the pleiotropic adipokine Leptin.
Authors: Alexandra Tsirigotaki / Ann Dansercoer / Koen H G Verschueren / Iva Marković / Christoph Pollmann / Maximillian Hafer / Jan Felix / Catherine Birck / Wouter Van Putte / Dominiek Catteeuw / ...Authors: Alexandra Tsirigotaki / Ann Dansercoer / Koen H G Verschueren / Iva Marković / Christoph Pollmann / Maximillian Hafer / Jan Felix / Catherine Birck / Wouter Van Putte / Dominiek Catteeuw / Jan Tavernier / J Fernando Bazan / Jacob Piehler / Savvas N Savvides / Kenneth Verstraete /
Abstract: The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and ...The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.
History
DepositionAug 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Leptin
B: Leptin receptor
C: Leptin
D: Leptin receptor
E: Leptin
F: Leptin receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)349,2349
Polymers349,0586
Non-polymers1763
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, The molecular weight as determined by SEC-MALLS corresponds to a 3:3 leptin-LEPR assembly.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Leptin / Obesity factor


Mass: 18873.283 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Mouse leptin was produced with an N-terminal His-tag and refolded from inclusion bodies produced in E. coli
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lep, Ob / Plasmid: pET15b / Details (production host): pET15b-His-TEV-mLeptin / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P41160
#2: Protein Leptin receptor / LEP-R / B219 / OB receptor / OB-R


Mass: 97479.391 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: The mLEP-R ectodomain was C-terminally fused to a trimeric GCN4 isoleucine zipper tag and secreted from HEK93 FreeStyle cells and complexed with refolded mouse leptin produced in E.coli.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lepr, Db, Obr / Plasmid: pTwist / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): FreeStyle / References: UniProt: P48356
#3: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ni
Has ligand of interestN

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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: Stucture of mouse leptin in complex with a trimerized form of the mouse Lep-R extracellular region
Type: COMPLEX
Details: The mLEP-R ectodomain was C-terminally fused to a trimeric GCN4 isoleucine zipper tag and secreted from HEK93 FreeStyle cells and complexed with refolded mouse leptin produced in E.coli.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.444 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: 293 Freestyle / Plasmid: pTwist
Buffer solutionpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 295 K

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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: FLOOD BEAM
Electron lensMode: OTHER / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13230

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selection
4cryoSPARCv3.3.1CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.19.2-4158model refinement
10cryoSPARCv3.3.1initial Euler assignment
11cryoSPARCv3.3.1final Euler assignment
12cryoSPARCv3.3.1classificationAb initio 3D classification
13cryoSPARCv3.3.13D reconstructionLocal refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 141157
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54708
Details: The final map was obtained via local refinement in cryoSPARC using a mask around the mouse leptin:LEP-R core region of the trimeric complex
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7Z3R

7z3r

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 240.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004813068
ELECTRON MICROSCOPYf_angle_d1.09517814
ELECTRON MICROSCOPYf_chiral_restr0.06382067
ELECTRON MICROSCOPYf_plane_restr0.00612259
ELECTRON MICROSCOPYf_dihedral_angle_d5.28161740

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