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- EMDB-29644: Structure of signaling thrombopoietin-MPL receptor complex -

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Basic information

Entry
Database: EMDB / ID: EMD-29644
TitleStructure of signaling thrombopoietin-MPL receptor complex
Map data
Sample
  • Complex: Thrombopoietin signaling complex with two thrombopoietin receptor chains
    • Protein or peptide: Thrombopoietin
    • Protein or peptide: Thrombopoietin receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose
KeywordsTPO / TpoR / cytokine / receptor / signaling / haematology
Function / homology
Function and homology information


thrombopoietin receptor activity / basophil homeostasis / monocyte homeostasis / thrombopoietin-mediated signaling pathway / positive regulation of hematopoietic stem cell proliferation / megakaryocyte differentiation / positive regulation of lymphocyte proliferation / cell surface receptor signaling pathway via STAT / positive regulation of platelet formation / positive regulation of megakaryocyte differentiation ...thrombopoietin receptor activity / basophil homeostasis / monocyte homeostasis / thrombopoietin-mediated signaling pathway / positive regulation of hematopoietic stem cell proliferation / megakaryocyte differentiation / positive regulation of lymphocyte proliferation / cell surface receptor signaling pathway via STAT / positive regulation of platelet formation / positive regulation of megakaryocyte differentiation / eosinophil homeostasis / neutrophil homeostasis / megakaryocyte development / Platelet Aggregation (Plug Formation) / immunoglobulin mediated immune response / cytokine activity / growth factor activity / hormone activity / cellular response to hypoxia / nuclear membrane / cell population proliferation / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of protein phosphorylation / external side of plasma membrane / signaling receptor binding / neuronal cell body / positive regulation of cell population proliferation / 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 / Four-helical cytokine-like, core ...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 / 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
Thrombopoietin / Thrombopoietin receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsTsutsumi N / Jude KM / Gati C / Garcia KC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R37AI051321 United States
Howard Hughes Medical Institute (HHMI) United States
Ludwig Institute for Cancer Research (LICR) United States
CitationJournal: Cell / Year: 2023
Title: Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.
Authors: Naotaka Tsutsumi / Zahra Masoumi / Sophie C James / Julie A Tucker / Hauke Winkelmann / William Grey / Lora K Picton / Lucie Moss / Steven C Wilson / Nathanael A Caveney / Kevin M Jude / ...Authors: Naotaka Tsutsumi / Zahra Masoumi / Sophie C James / Julie A Tucker / Hauke Winkelmann / William Grey / Lora K Picton / Lucie Moss / Steven C Wilson / Nathanael A Caveney / Kevin M Jude / Cornelius Gati / Jacob Piehler / Ian S Hitchcock / K Christopher Garcia /
Abstract: Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy ...Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPO) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPO decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPO in hematology research and clinical HSC transplantation.
History
DepositionJan 31, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29644.png

Downloads & links

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Map

FileDownload / File: emd_29644.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 300 pix.
= 323.4 Å		1.08 Å/pix.
x 300 pix.
= 323.4 Å		1.08 Å/pix.
x 300 pix.
= 323.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.078 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-1.8591155 - 3.752217
Average (Standard dev.)0.0005373931 (±0.04246191)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 323.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29644_msk_1.map
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Mask #2

Fileemd_29644_msk_2.map
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Additional map: deepEMhancer sharpened map

Fileemd_29644_additional_1.map
AnnotationdeepEMhancer sharpened map
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Additional map: unsharpen map

Fileemd_29644_additional_2.map
Annotationunsharpen map
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Half map: #2

Fileemd_29644_half_map_1.map
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Half map: #1

Fileemd_29644_half_map_2.map
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Sample components

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Entire : Thrombopoietin signaling complex with two thrombopoietin receptor...

EntireName: Thrombopoietin signaling complex with two thrombopoietin receptor chains
Components
  • Complex: Thrombopoietin signaling complex with two thrombopoietin receptor chains
    • Protein or peptide: Thrombopoietin
    • Protein or peptide: Thrombopoietin receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose

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Supramolecule #1: Thrombopoietin signaling complex with two thrombopoietin receptor...

SupramoleculeName: Thrombopoietin signaling complex with two thrombopoietin receptor chains
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: Thrombopoietin

MacromoleculeName: Thrombopoietin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.381322 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
DYKDDDDGSP APPACDLRVL SKLLRDSHVL HSRLSQCPEV HPLPTPVLLP AVDFSLGEWK TQMEETKAQD ILGAVTLLLE GVMAARGQL GPTCLSSLLG QLSGQVRLLL GALQSLLGTQ LPPQGRTTAH KDPNAIFLSF QHLLRGKVRF LMLVGGSTLC V RRAPPTTA VPS

UniProtKB: Thrombopoietin

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Macromolecule #2: Thrombopoietin receptor

MacromoleculeName: Thrombopoietin receptor / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 71.493445 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: (PCA)DVSLLASDS EPLKCFSRTF EDLTCFWDEE EAAPSGTYQL LYAYPREKPR ACPLSSQSMP HFGTRYVCQF PDQEEV RLF FPLHLWVKNV FLNQTRTQRV LFVDSVGLPA PPSIIKAMGG SQPGELQISW EEPAPEISDF LRYELRYGPR DPKNSTG PT VIQLIATETC ...String:
(PCA)DVSLLASDS EPLKCFSRTF EDLTCFWDEE EAAPSGTYQL LYAYPREKPR ACPLSSQSMP HFGTRYVCQF PDQEEV RLF FPLHLWVKNV FLNQTRTQRV LFVDSVGLPA PPSIIKAMGG SQPGELQISW EEPAPEISDF LRYELRYGPR DPKNSTG PT VIQLIATETC CPALQRPHSA SALDQSPCAQ PTMPWQDGPK QTSPSREASA LTAEGGSCLI SGLQPGNSYW LQLRSEPD G ISLGGSWGSW SLPVTVDLPG DAVALGLQCF TLDLKNVTCQ WQQQDHASSQ GFFYHSRARC CPRDRYPIWE NCEEEEKTN PGLQTPQFSR CHFKSRNDSI IHILVEVTTA PGTVHSYLGS PFWIHQAVRL PTPNLHWREI SSGHLELEWQ HPSSWAAQET CYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALHL V LGLSAVLG LLLLRWQFPA HYRRLRHALW PSLPDLHRVL GQYLRDTAAL SPPKATVSDT CEEVEPSLLE ILPKSSERTP LP LCSSQAQ MDYRRLQPSC LGTMPLSVCP PMAESGSCCT THIANHSYLP LSYWQQPAAA GAAEDQVDPR LIDGKHHHHH HHH

UniProtKB: Thrombopoietin receptor

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #4: alpha-D-mannopyranose

MacromoleculeName: alpha-D-mannopyranose / type: ligand / ID: 4 / Number of copies: 6 / 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

Concentration3 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
10.0 mMHEPES-Na
150.0 mMSodium chloride
0.001 % (w/v)LMNG
0.05 % (w/v)Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 281 K / Instrument: LEICA EM GP / Details: 3s blotting.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 10494 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 46382 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 9537683
Startup modelType of model: OTHER / Details: Initial model generation in cryoSPARC.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 329658
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1v7n


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8g04:
Structure of signaling thrombopoietin-MPL receptor complex

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