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- PDB-8b7q: Cryo-EM structure for the mouse LEPR-CRH2:Leptin:LEPR-Ig complex ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8b7q | ||||||
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Title | Cryo-EM structure for the mouse LEPR-CRH2:Leptin:LEPR-Ig complex following symmetry expansion in combination with local refinement | ||||||
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![]() | CYTOKINE / leptin / LEP-R / obesity / metabolism / energy balance | ||||||
Function / homology | ![]() 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 / central nervous system neuron development / fatty acid beta-oxidation / 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 / positive regulation of insulin receptor signaling pathway / cellular response to retinoic acid / positive regulation of T cell proliferation / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of interleukin-12 production / cholesterol metabolic process / negative regulation of autophagy / response to activity / positive regulation of interleukin-8 production / gluconeogenesis / determination of adult lifespan / female pregnancy / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein phosphorylation / placenta development / response to insulin Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å | ||||||
![]() | Verstraete, K. / Savvides, S.N. / Verschueren, K.G. / Tsirigotaki, A. | ||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 148.4 KB | Display | ![]() |
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PDB format | ![]() | 93.2 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 1.4 MB | Display | |
Data in XML | ![]() | 36.6 KB | Display | |
Data in CIF | ![]() | 50.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 15899MC ![]() 7z3pC ![]() 7z3qC ![]() 7z3rC ![]() 8av2C ![]() 8avbC ![]() 8avcC ![]() 8avdC ![]() 8aveC ![]() 8avfC ![]() 8avoC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 18873.283 Da / Num. of mol.: 1 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.) ![]() ![]() ![]() ![]() | ||||
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#2: Protein | Mass: 97479.391 Da / Num. of mol.: 2 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.) ![]() ![]() ![]() #3: Sugar | ChemComp-NAG / | Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: 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 | |||||||||||||||
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Molecular weight | Value: 0.444 MDa / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||
Source (recombinant) | Organism: ![]() | |||||||||||||||
Buffer solution | pH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, pH 7.4 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse. | |||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 | |||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: OTHER / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm |
Image recording | Electron 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
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 141157 | |||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163914 Details: To address the breakage of symmetry in the ring-like core region of the complex and possibly revolve the mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex at higher resolution, the pseudo-C3 ...Details: To address the breakage of symmetry in the ring-like core region of the complex and possibly revolve the mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex at higher resolution, the pseudo-C3 symmetric volume following consenus refinement was aligned to the pseudo-C3 symmetry axis via the Volume Alignment Tool job in cryoSPARC. The associated particle set was re-extracted without binning and symmetry expanded around the C3 axis resulting in 163,914 particles. Using the molmap function in Chimera, a volume blurred to 25 Angstrom around one mLEP-RCRH2:mLeptin:mLEP-RCRH1-IgCRH2 subcomplex was generated, and transformed into a mask with the Volume Tools job in cryoSPARC. Local refinement was performed by limiting the rotation and shift search extent around the original consensus refinement. The center of mass of the mask was used as a fulcrum point. This approach resulted in a cryo-EM map with an FSC0.143 resolution of 4.02 Angstrom in which the crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex was fitted using Chimera and real-space refined in Phenix using reference restraints to the starting model. Symmetry type: POINT | |||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL Details: The crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex (pdb 7z3r) was fitted in the cryo-EM map using Chimera and real-space refined in Phenix using reference ...Details: The crystallographic model for the mLEP-RCRH2:mLeptin:mLEP-R_IgCRH2' complex (pdb 7z3r) was fitted in the cryo-EM map using Chimera and real-space refined in Phenix using reference restraints to the starting model. | |||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 101.59 Å2 | |||||||||||||||||||||||||||||||||||
Refine LS restraints |
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