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- EMDB-41805: Cryo-EM structure of murine Thrombopoietin receptor ectodomain in... -

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Entry
Database: EMDB / ID: EMD-41805
TitleCryo-EM structure of murine Thrombopoietin receptor ectodomain in complex with Tpo
Map dataRefinement map from 3Dflex after non-uniform refinement. Sharpened with deepEMhancer
Sample
  • Complex: 2:1 complex of the mouse thrombopoietin receptor ectodomain and mouse thrombopoietin
    • Complex: mouse thrombopoietin receptor ectodomain
      • Protein or peptide: Thrombopoietin receptor,GCN4 isoform 1
    • Complex: mouse thrombopoietin
      • Protein or peptide: Thrombopoietin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose
KeywordsReceptor / 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
GCN4 isoform 1 / Thrombopoietin / Thrombopoietin receptor
Similarity search - Component
Biological speciesMus musculus (house mouse) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSarson-Lawrence KS / Hardy JM / Leis A / Babon JJ / Kershaw NJ
Funding support Australia, 2 items
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, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41805.png

Downloads & links

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Map

FileDownload / File: emd_41805.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefinement map from 3Dflex after non-uniform refinement. Sharpened with deepEMhancer
Voxel sizeX=Y=Z: 1.02626 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.46230578 - 2.362889
Average (Standard dev.)0.006190967 (±0.06089593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 205.25119 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 3DFlex Refinement Map

Fileemd_41805_additional_1.map
Annotation3DFlex Refinement Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3DFlex Half Map B

Fileemd_41805_half_map_1.map
Annotation3DFlex Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3DFlex Half Map A

Fileemd_41805_half_map_2.map
Annotation3DFlex Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 2:1 complex of the mouse thrombopoietin receptor ectodomain and m...

EntireName: 2:1 complex of the mouse thrombopoietin receptor ectodomain and mouse thrombopoietin
Components
  • Complex: 2:1 complex of the mouse thrombopoietin receptor ectodomain and mouse thrombopoietin
    • Complex: mouse thrombopoietin receptor ectodomain
      • Protein or peptide: Thrombopoietin receptor,GCN4 isoform 1
    • Complex: mouse thrombopoietin
      • Protein or peptide: Thrombopoietin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose

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Supramolecule #1: 2:1 complex of the mouse thrombopoietin receptor ectodomain and m...

SupramoleculeName: 2:1 complex of the mouse thrombopoietin receptor ectodomain and mouse thrombopoietin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: mTpoR 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 ...Details: mTpoR 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.
Molecular weightTheoretical: 131.698 KDa

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Supramolecule #2: mouse thrombopoietin receptor ectodomain

SupramoleculeName: mouse thrombopoietin receptor ectodomain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: mouse thrombopoietin

SupramoleculeName: mouse thrombopoietin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Thrombopoietin receptor,GCN4 isoform 1

MacromoleculeName: Thrombopoietin receptor,GCN4 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 56.559676 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QDVFLLALGT EPLNCFSQTF EDLTCFWDEE EAAPSGTYQL LYAYRGEKPR ACPLYSQSVP TFGTRYVCQF PAQDEVRLFF PLHLWVKNV SLNQTLIQRV LFVDSVGLPA PPRVIKARGG SQPGELQIHW EAPAPEISDF LRHELRYGPT DSSNATAPSV I QLLSTETC ...String:
QDVFLLALGT EPLNCFSQTF EDLTCFWDEE EAAPSGTYQL LYAYRGEKPR ACPLYSQSVP TFGTRYVCQF PAQDEVRLFF PLHLWVKNV SLNQTLIQRV LFVDSVGLPA PPRVIKARGG SQPGELQIHW EAPAPEISDF LRHELRYGPT DSSNATAPSV I QLLSTETC CPTLWMPNPV PVLDQPPCVH PTASQPHGPA PFLTVKGGSC LVSGLQAGKS YWLQLRSQPD GVSLRGSWGP WS FPVTVDL PGDAVTIGLQ CFTLDLKMVT CQWQQQDRTS SQGFFRHSRT RCCPTDRDPT WEKCEEEEPR PGSQPALVSR CHF KSRNDS VIHILVEVTT AQGAVHSYLG SPFWIHQAVL LPTPSLHWRE VSSGRLELEW QHQSSWAAQE TCYQLRYTGE GRED WKVLE PSLGARGGTL ELRPRARYSL QLRARLNGPT YQGPWSAWSP PARVSTGSET AWRMKQLEDK VEELLSKNYH LENEV ARLK KLVGERTGTG GAPENLYFQ

UniProtKB: Thrombopoietin receptor, GCN4 isoform 1

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Macromolecule #2: Thrombopoietin

MacromoleculeName: Thrombopoietin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 19.611674 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ASISARQDYK DDDDKTRQSP VAPACDPRLL NKLLRDSHLL HSRLSQCPDV DPLSIPVLLP AVDFSLGEWK TQTEQSKAQD ILGAVSLLL EGVMAARGQL EPSCLSSLLG QLSGQVRLLL GALQGLLGTQ LPLQGRTTAH KDPNALFLSL QQLLRGKVRF L LLVEGPTL CVRRTLPTTA VPS

UniProtKB: Thrombopoietin

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: alpha-D-mannopyranose

MacromoleculeName: alpha-D-mannopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: MAN
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-MAN:
alpha-D-mannopyranose

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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

Concentration0.55 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
50.0 mMTris-HCLtris(hydroxymethyl)aminomethane - hydrochloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time of 4 s, blot force of 6 and no wait time or drain time..
DetailsTpoR1-479-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).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6130 / Average exposure time: 2.38 sec. / Average electron dose: 50.57 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies 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.
Particle selectionNumber 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.
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC
Final reconstructionNumber classes used: 73 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1)
Details: Final map was generated using Non-uniform refinement followed by 3DFlex refinement in cryoSPARC. Map was sharpened by deepEMhancer.
Number images used: 745000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1) / Details: Non-uniform refinement in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1) / Details: Non-uniform refinement in cryoSPARC
Final 3D classificationNumber classes: 100 / Avg.num./class: 9146 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: Multimer complex of the complete assembly was produced using AlphaFold2 on Google CoLab
DetailsChimeraX was used to perform rigid-body fitting of domains and modelling was performed using Coot and ISOLDE. Refinement was carried out in PHENIX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8u18:
Cryo-EM structure of murine Thrombopoietin receptor ectodomain in complex with Tpo

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