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- PDB-8u18: Cryo-EM structure of murine Thrombopoietin receptor ectodomain in... -

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Basic information

Entry
Database: PDB / ID: 8u18
TitleCryo-EM structure of murine Thrombopoietin receptor ectodomain in complex with Tpo
Components
  • Thrombopoietin
  • Thrombopoietin receptor,GCN4 isoform 1
KeywordsCYTOKINE/RECEPTOR / Receptor / cytokine / signalling / haematopoiesis / CYTOKINE-RECEPTOR complex
Function / homology
Function and homology information


thrombopoietin receptor activity / basophil homeostasis / regulation of chemokine production / monocyte homeostasis / thrombopoietin-mediated signaling pathway / positive regulation of hematopoietic stem cell proliferation / cell surface receptor signaling pathway via STAT / regulation of stem cell division / positive regulation of lymphocyte proliferation / megakaryocyte differentiation ...thrombopoietin receptor activity / basophil homeostasis / regulation of chemokine production / monocyte homeostasis / thrombopoietin-mediated signaling pathway / positive regulation of hematopoietic stem cell proliferation / cell surface receptor signaling pathway via STAT / regulation of stem cell division / positive regulation of lymphocyte proliferation / megakaryocyte differentiation / positive regulation of megakaryocyte differentiation / positive regulation of platelet formation / eosinophil homeostasis / regulation of stem cell proliferation / myeloid cell differentiation / neutrophil homeostasis / definitive hemopoiesis / platelet formation / megakaryocyte development / homeostasis of number of cells / immunoglobulin mediated immune response / cytokine activity / hormone activity / nuclear membrane / cell population proliferation / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of ERK1 and ERK2 cascade / positive regulation of protein phosphorylation / DNA-binding transcription factor activity / external side of plasma membrane / signaling receptor binding / neuronal cell body / positive regulation of cell population proliferation / positive regulation of gene expression / negative regulation of apoptotic process / Golgi apparatus / cell surface / extracellular space / extracellular region / plasma membrane
Similarity search - Function
Thrombopoietin / Erythropoietin/thrombopoietin / Erythropoietin/thrombopoeitin, conserved site / Erythropoietin/thrombopoietin / Erythropoietin / thrombopoeitin signature. / Long hematopoietin receptor, single chain, conserved site / Long hematopoietin receptor, single chain family signature. / Growth hormone/erythropoietin receptor, ligand binding / Erythropoietin receptor, ligand binding / Basic region leucine zipper ...Thrombopoietin / Erythropoietin/thrombopoietin / Erythropoietin/thrombopoeitin, conserved site / Erythropoietin/thrombopoietin / Erythropoietin / thrombopoeitin signature. / Long hematopoietin receptor, single chain, conserved site / Long hematopoietin receptor, single chain family signature. / Growth hormone/erythropoietin receptor, ligand binding / Erythropoietin receptor, ligand binding / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / Four-helical cytokine-like, core / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
alpha-D-mannopyranose / GCN4 isoform 1 / Thrombopoietin / Thrombopoietin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSarson-Lawrence, K.S. / Hardy, J.M. / Leis, A. / Babon, J.J. / Kershaw, N.J.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)2008096 Australia
The Walter and Eliza Hall Institute of Medical Research Australia
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin.
Authors: Kaiseal T G Sarson-Lawrence / Joshua M Hardy / Josephine Iaria / Dina Stockwell / Kira Behrens / Tamanna Saiyed / Cyrus Tan / Leila Jebeli / Nichollas E Scott / Toby A Dite / Nicos A Nicola ...Authors: Kaiseal T G Sarson-Lawrence / Joshua M Hardy / Josephine Iaria / Dina Stockwell / Kira Behrens / Tamanna Saiyed / Cyrus Tan / Leila Jebeli / Nichollas E Scott / Toby A Dite / Nicos A Nicola / Andrew P Leis / Jeffrey J Babon / Nadia J Kershaw /
Abstract: Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric ...Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists.
History
DepositionAug 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thrombopoietin receptor,GCN4 isoform 1
B: Thrombopoietin receptor,GCN4 isoform 1
C: Thrombopoietin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,14911
Polymers132,7313
Non-polymers2,4188
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration, SEC-MALLS confirmed size and ratio of complex, light scattering, Mass photometry confirmed complex size
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Thrombopoietin receptor,GCN4 isoform 1 / TPO-R / Myeloproliferative leukemia protein / Proto-oncogene c-Mpl


Mass: 56559.676 Da / Num. of mol.: 2
Fragment: extracellular domain,leucine zipper,extracellular domain,leucine zipper
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: Mpl, Tpor, GCN4 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q08351, UniProt: A0A8H8ULK5
#2: Protein Thrombopoietin / C-mpl ligand / ML / Megakaryocyte colony-stimulating factor / Megakaryocyte growth and development ...C-mpl ligand / ML / Megakaryocyte colony-stimulating factor / Megakaryocyte growth and development factor / MGDF / Myeloproliferative leukemia virus oncogene ligand


Mass: 19611.674 Da / Num. of mol.: 1 / Fragment: N-terminal domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Thpo / Cell line (production host): FreeStyle 293-F / Production host: Homo sapiens (human) / References: UniProt: P40226
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Sugar ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
12:1 complex of the mouse thrombopoietin receptor ectodomain and mouse thrombopoietinCOMPLEXmTpoR was expressed as a fusion of residues 1-482 of mTPOR, followed by a GCN4 leucine zipper from Saccharomyces cerevisiae, a TEV protease site, the Fc domain of hlgG1, and a C-terminal FLAG tag. The mTpoR signal peptide (1-25) was cleaved during expression and the Fc tag was cleaved by TEV before complexation. mTPO was expressed as a fusion of the mIL3 secretion signal (cleaved during expression), an N-terminal FLAG-tag followed by residues 22 to 184 of mTPO.#1-#20MULTIPLE SOURCES
2mouse thrombopoietin receptor ectodomainCOMPLEX#11RECOMBINANT
3mouse thrombopoietinCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.131698 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
32Saccharomyces cerevisiae (brewer's yeast)4932
43Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606Expi293F
33Homo sapiens (human)9606Expi293F
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
250 mMtris(hydroxymethyl)aminomethane - hydrochlorideTris-HCL1
SpecimenConc.: 0.55 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: TpoR1-479-LeuZ and TPON were combined with a molar ratio of 1:1.5 and applied to superdex 200 10/300 increased column equilibrated in TBS pH7.5. The resultant peaks were analysed by SDS-PAGE ...Details: TpoR1-479-LeuZ and TPON were combined with a molar ratio of 1:1.5 and applied to superdex 200 10/300 increased column equilibrated in TBS pH7.5. The resultant peaks were analysed by SDS-PAGE and SEC-MALS, and those corresponding to the complex were pooled and concentrated to 0.55 mg/ml. CTAB (Hexadecyl-trimethylammonium bromide) was added right before plunge freezing to a concentration of 0.005% (w/v).
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K
Details: Blot time of 4 s, blot force of 6 and no wait time or drain time.

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.38 sec. / Electron dose: 50.57 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6130
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2EPU2image acquisition
4cryoSPARC4.2.1CTF correction
7ISOLDE1.6.0model fitting
8UCSF ChimeraX1.6.1model fitting
9Coot0.9.8.3model fitting
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.1classification
14cryoSPARC4.2.13D reconstruction
15PHENIX1.20.1_4487model refinement
Image processingDetails: Movies were aligned using patch motion correction and defocus values were estimated using patch CTF estimation. Images with significant astigmatism, ice contamination, or drift were removed ...Details: Movies were aligned using patch motion correction and defocus values were estimated using patch CTF estimation. Images with significant astigmatism, ice contamination, or drift were removed resulting in 5,297 micrographs.
CTF correctionDetails: Defocus values were estimated using patch CTF estimation and CTF correction was performed during 3D reccnstruction.
Type: NONE
Particle selectionNum. of particles selected: 2353523
Details: Templates for picking were generated using a gaussian based picking approach, followed by 2D classification and selection of high-quality 2D classes for template picking.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 745000 / Algorithm: BACK PROJECTION
Details: Final map was generated using Non-uniform refinement followed by 3DFlex refinement in cryoSPARC. Map was sharpened by deepEMhancer.
Num. of class averages: 73 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: ChimeraX was used to perform rigid-body fitting of domains and modelling was performed using Coot and ISOLDE. Refinement was carried out in PHENIX.
Atomic model buildingDetails: Multimer complex of the complete assembly was produced using AlphaFold2 on Google CoLab
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048258
ELECTRON MICROSCOPYf_angle_d0.72811304
ELECTRON MICROSCOPYf_dihedral_angle_d9.941181
ELECTRON MICROSCOPYf_chiral_restr0.0441274
ELECTRON MICROSCOPYf_plane_restr0.0061457

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