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

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Basic information

Entry
Database: PDB / ID: 9cth
TitlePreliminary map of the Prothrombin-prothrombinase complex on nano discs
Components
  • (Activated Factor V (FVa) ...) x 2
  • (Activated Factor X ...) x 2
  • Prothrombin
KeywordsBLOOD CLOTTING / Coagulation / Prothrombin / Prothrombinase / nanodisc / complex
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 / Extrinsic Pathway of Fibrin Clot Formation / COPII-mediated vesicle transport / blood circulation ...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 / Extrinsic Pathway of Fibrin Clot Formation / COPII-mediated vesicle transport / blood circulation / cytolysis by host of symbiont cells / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin-activated receptor signaling pathway / thrombin / COPII-coated ER to Golgi transport vesicle / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / Defective F8 cleavage by thrombin / ligand-gated ion channel signaling pathway / Platelet Aggregation (Plug Formation) / negative regulation of astrocyte differentiation / positive regulation of collagen biosynthetic process / negative regulation of platelet activation / negative regulation of blood coagulation / positive regulation of blood coagulation / positive regulation of TOR signaling / negative regulation of fibrinolysis / 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 / negative regulation of proteolysis / Intrinsic Pathway of Fibrin Clot Formation / negative regulation of cytokine production involved in inflammatory response / endoplasmic reticulum-Golgi intermediate compartment membrane / 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 / positive regulation of receptor signaling pathway via JAK-STAT / Cell surface interactions at the vascular wall / Post-translational protein phosphorylation / lipopolysaccharide binding / growth factor activity / positive regulation of insulin secretion / phospholipid binding / platelet activation / positive regulation of protein localization to nucleus / response to wounding / Golgi lumen / antimicrobial humoral immune response mediated by antimicrobial peptide / 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 / Platelet degranulation / extracellular vesicle / heparin binding / regulation of cell shape / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of protein phosphorylation / positive regulation of cell growth / : / G alpha (q) signalling events / blood microparticle / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell surface receptor signaling pathway / positive regulation of cell migration / receptor ligand activity / copper ion binding / endoplasmic reticulum lumen / external side of plasma membrane / signaling receptor binding / serine-type endopeptidase activity / 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. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.47 Å
AuthorsStojanovski, B.M. / Mohammed, B.M. / Di Cera, E.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL049413, HL139554, HL147821 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 United States
Other privateThe Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine CA091842 United States
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
DepositionJul 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
SupersessionAug 14, 2024ID: 7TPQ
Revision 1.1Aug 14, 2024Group: Advisory / Category: pdbx_database_PDB_obs_spr
Revision 1.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Activated Factor V (FVa) heavy chain
E: Activated Factor V (FVa) light chain
B: Activated Factor X light chain
C: Activated Factor X heavy chain
D: Prothrombin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)266,03011
Polymers264,7035
Non-polymers1,3276
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Activated Factor V (FVa) ... , 2 types, 2 molecules AE

#1: Protein Activated Factor V (FVa) heavy chain / Coagulation factor Va heavy chain / Activated protein C cofactor / Proaccelerin / labile factor


Mass: 81274.391 Da / Num. of mol.: 1 / Fragment: Domains A1 and A2 (UNP residues 29-737) / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Tissue: Blood / References: UniProt: P12259
#2: Protein Activated Factor V (FVa) light chain / Coagulation factor Va light chain / Activated protein C cofactor / Proaccelerin / labile factor


Mass: 75283.008 Da / Num. of mol.: 1 / Fragment: Domains C1, C2, and A3 (UNP residues 1574-2224) / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Tissue: Blood / References: UniProt: P12259

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Activated Factor X ... , 2 types, 2 molecules BC

#3: Protein Activated Factor X light chain / FXa-LC / Factor X light chain


Mass: 16186.952 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F10 / Cell (production host): fibroblasts / Cell line (production host): BHK / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00742
#4: Protein Activated Factor X heavy chain / FXa-HC / Factor X heavy chain


Mass: 26588.297 Da / Num. of mol.: 1 / Mutation: S195A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F10 / Cell (production host): fibroblasts / Cell line (production host): BHK / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00742

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Protein / Sugars , 2 types, 7 molecules D

#5: Protein Prothrombin / Coagulation factor II


Mass: 65370.113 Da / Num. of mol.: 1 / Fragment: UNP residues 44-622 / Mutation: S525A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): fibroblasts / Cell line (production host): BHK / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00734, thrombin
#6: 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.: 6 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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

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Details

Has ligand of interestY
Has protein modificationY

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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

ComponentName: Prothrombinase:Prothrombin ternary complex on lipid nanodiscs
Type: COMPLEX
Details: The complex is made of coagulation factor Va (derived from human plasma), active site mutated (S195A) coagulation factor Xa (Recombinantly expressed in BHK cells) and active site mutated ...Details: The complex is made of coagulation factor Va (derived from human plasma), active site mutated (S195A) coagulation factor Xa (Recombinantly expressed in BHK cells) and active site mutated (S525A) 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.
Entity ID: #3-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mesocricetus auratus (golden hamster)
Buffer solutionpH: 7.4 / Details: 20 mM HEPES, 150 mM NaCl, and 5 mM CaCl2
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Prothrombin:FVa:FXa were mixed in 2:1:2 ratio. 0.01 mg/mL total protein was used for freezing.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 51.28 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 2390

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.1particle selection
2EPUimage acquisition
4cryoSPARC4.3.1CTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
12cryoSPARC4.3.1classification
13cryoSPARC4.3.13D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4988 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18TN9

8tn9

18TN91PDBexperimental model
21AF-P00734-F12AlphaFoldin silico model
31AF-P00742-F13AlphaFoldin silico model
41fjs

1fjs

11fjs4PDBexperimental model
56bjr

6bjr

16bjr5PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318590
ELECTRON MICROSCOPYf_angle_d0.66225151
ELECTRON MICROSCOPYf_dihedral_angle_d6.0512465
ELECTRON MICROSCOPYf_chiral_restr0.052658
ELECTRON MICROSCOPYf_plane_restr0.0043267

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