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- EMDB-42405: Preliminary map of the Prothrombin-prothrombinase complex on nano... -

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Basic information

Entry
Database: EMDB / ID: EMD-42405
TitlePreliminary map of the Prothrombin-prothrombinase complex on nano discs
Map dataUnsharpened map
Sample
  • Complex: Prothrombin_Prothrombinase complex on lipid nanodiscs.
    • Organelle or cellular component: Coagulation Factor Va
    • Organelle or cellular component: Coagulation Factor Xa
    • Organelle or cellular component: Prothrombin
KeywordsCoagulation / Prothrombin / Prothrombinase / nanodisc / complex / BLOOD CLOTTING
Function / homology
Function and homology information


response to vitamin K / coagulation factor Xa / platelet alpha granule / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-coated ER to Golgi transport vesicle / Extrinsic Pathway of Fibrin Clot Formation / COPII-mediated vesicle transport ...response to vitamin K / coagulation factor Xa / platelet alpha granule / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-coated ER to Golgi transport vesicle / Extrinsic Pathway of Fibrin Clot Formation / COPII-mediated vesicle transport / blood circulation / cytolysis by host of symbiont cells / thrombospondin receptor activity / thrombin-activated receptor signaling pathway / Defective factor XII causes hereditary angioedema / thrombin / negative regulation of astrocyte differentiation / regulation of blood coagulation / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / neutrophil-mediated killing of gram-negative bacterium / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / ligand-gated ion channel signaling pathway / positive regulation of collagen biosynthetic process / negative regulation of platelet activation / negative regulation of blood coagulation / positive regulation of blood coagulation / negative regulation of fibrinolysis / positive regulation of TOR signaling / regulation of cytosolic calcium ion concentration / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Gamma-carboxylation of protein precursors / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / negative regulation of proteolysis / endoplasmic reticulum-Golgi intermediate compartment membrane / negative regulation of cytokine production involved in inflammatory response / positive regulation of release of sequestered calcium ion into cytosol / Peptide ligand-binding receptors / platelet alpha granule lumen / Regulation of Complement cascade / acute-phase response / Cell surface interactions at the vascular wall / positive regulation of receptor signaling pathway via JAK-STAT / Post-translational protein phosphorylation / lipopolysaccharide binding / growth factor activity / phospholipid binding / positive regulation of insulin secretion / platelet activation / response to wounding / Golgi lumen / positive regulation of protein localization to nucleus / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / positive regulation of reactive oxygen species metabolic process / blood coagulation / antimicrobial humoral immune response mediated by antimicrobial peptide / Platelet degranulation / extracellular vesicle / regulation of cell shape / heparin binding / Thrombin signalling through proteinase activated receptors (PARs) / : / positive regulation of cell growth / blood microparticle / G alpha (q) signalling events / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cell migration / receptor ligand activity / endoplasmic reticulum lumen / copper ion binding / signaling receptor binding / serine-type endopeptidase activity / external side of plasma membrane / positive regulation of cell population proliferation / calcium ion binding / proteolysis / extracellular space / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : ...Coagulation factor 5/8-like / : / Coagulation factors 5/8 type C domain (FA58C) signature 2. / Multicopper oxidases, conserved site / Multicopper oxidases signature 1. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : / Coagulation factor-like, Gla domain superfamily / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / : / Thrombin light chain / Coagulation Factor Xa inhibitory site / Multicopper oxidase, N-terminal / Multicopper oxidase / EGF-like domain / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / Kringle-like fold / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / Cupredoxin / EGF-like domain / Galactose-binding-like domain superfamily / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Prothrombin / Coagulation factor X / Coagulation factor V
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.47 Å
AuthorsStojanovski BM / Mohammed BM / Di Cera E
Funding support United States, 8 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL049413 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL139554 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL147821 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalCDI-CORE-2015-505 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalCDI-CORE-2019-813 United States
Foundation for Barnes-Jewish Hospital3770 United States
Other privateWashington University Diabetes Research Center DK020579
Other privateThe Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine CA091842
CitationJournal: Subcell Biochem / Year: 2024
Title: The Prothrombin-Prothrombinase Interaction.
Authors: Bosko M Stojanovski / Bassem M Mohammed / Enrico Di Cera /
Abstract: The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with ...The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with exposure of tissue factor from the damaged endothelium and culminates with conversion of prothrombin to thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca, and phospholipids. This cofactor-dependent activation is paradigmatic of analogous reactions of the blood coagulation and complement cascades, which makes elucidation of its molecular mechanism of broad significance to the large class of trypsin-like zymogens to which prothrombin belongs. Because of its relevance as the most important reaction in the physiological response to vascular injury, as well as the main trigger of pathological thrombotic complications, the mechanism of prothrombin activation has been studied extensively. However, a molecular interpretation of this mechanism has become available only recently from important developments in structural biology. Here we review current knowledge on the prothrombin-prothrombinase interaction and outline future directions for the study of this key reaction of the coagulation cascade.
History
DepositionOct 19, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_42405.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.0839
Minimum - Maximum-0.19871338 - 0.76784295
Average (Standard dev.)0.0016278322 (±0.028282389)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 481.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Prothrombin_Prothrombinase complex on lipid nanodiscs.

EntireName: Prothrombin_Prothrombinase complex on lipid nanodiscs.
Components
  • Complex: Prothrombin_Prothrombinase complex on lipid nanodiscs.
    • Organelle or cellular component: Coagulation Factor Va
    • Organelle or cellular component: Coagulation Factor Xa
    • Organelle or cellular component: Prothrombin

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Supramolecule #1: Prothrombin_Prothrombinase complex on lipid nanodiscs.

SupramoleculeName: Prothrombin_Prothrombinase complex on lipid nanodiscs.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The complex is made of coagulation factor Va (derived from human plasma), coagulation factor Xa (Recombinantly expressed in BHK cells) and Prothrombin (Recombinantly expressed in BHK cells). ...Details: The complex is made of coagulation factor Va (derived from human plasma), coagulation factor Xa (Recombinantly expressed in BHK cells) and Prothrombin (Recombinantly expressed in BHK cells). The nanodisc component of the complex is made of the scaffold protein MSP1E3D1 (Recombinantly expressed in bacteria) and the phospholipid component was Porcine Brain phosphatidylserine.
Source (natural)Organism: Homo sapiens (human) / Tissue: Blood

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Supramolecule #2: Coagulation Factor Va

SupramoleculeName: Coagulation Factor Va / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human) / Tissue: Blood

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Supramolecule #3: Coagulation Factor Xa

SupramoleculeName: Coagulation Factor Xa / type: organelle_or_cellular_component / ID: 3 / Parent: 1
Details: Catalytically inactive full length mutant S379A with C-terminus HPC4 tag.
Source (natural)Organism: Homo sapiens (human) / Tissue: Blood

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Supramolecule #4: Prothrombin

SupramoleculeName: Prothrombin / type: organelle_or_cellular_component / ID: 4 / Parent: 1
Details: Catalytically inactive full length mutant S525A with C-terminus HPC4 tag
Source (natural)Organism: Homo sapiens (human) / Tissue: Blood

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, and 5 mM CaCl2
GridMaterial: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsProthrombin:FVa:FXa were mixed in 2:1:2 ratio. 0.01 mg/mL total protein was used for freezing.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 2390 / Average electron dose: 51.28 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 4988
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 3000 / Software - Name: cryoSPARC (ver. 4.3.1)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-9cth:
Preliminary map of the Prothrombin-prothrombinase complex on nano discs

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