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- PDB-7tpp: Cryo-em structure of human prothrombin:prothrombinase at 4.1 Angs... -

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Basic information

Entry
Database: PDB / ID: 7tpp
TitleCryo-em structure of human prothrombin:prothrombinase at 4.1 Angstrom resolution
Components
  • (Coagulation factor Va) x 2
  • Activated factor Xa heavy chain
  • Factor X light chain
  • ProthrombinThrombin
KeywordsBLOOD CLOTTING / prothrombin prothrombinase
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 / blood circulation / 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 / blood circulation / Extrinsic Pathway of Fibrin Clot Formation / COPII-mediated vesicle transport / positive regulation of lipid kinase activity / COPII-coated ER to Golgi transport vesicle / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / negative regulation of astrocyte differentiation / positive regulation of TOR signaling / negative regulation of platelet activation / positive regulation of collagen biosynthetic process / negative regulation of cytokine production involved in inflammatory response / positive regulation of blood coagulation / negative regulation of fibrinolysis / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / regulation of cytosolic calcium ion concentration / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / platelet alpha granule lumen / Regulation of Complement cascade / acute-phase response / Cell surface interactions at the vascular wall / lipopolysaccharide binding / negative regulation of proteolysis / Post-translational protein phosphorylation / positive regulation of receptor signaling pathway via JAK-STAT / phospholipid binding / growth factor activity / 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 / extracellular vesicle / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / Thrombin signalling through proteinase activated receptors (PARs) / Platelet degranulation / heparin binding / regulation of cell shape / positive regulation of cell growth / G alpha (q) signalling events / collagen-containing extracellular matrix / blood microparticle / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cell migration / positive regulation of protein phosphorylation / copper ion binding / G protein-coupled receptor signaling pathway / external side of plasma membrane / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / calcium ion binding / positive regulation of cell population proliferation / 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-like, Gla domain superfamily / F5/8 type C domain ...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-type aspartate/asparagine hydroxylation site / EGF-like domain / 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. / Epidermal growth factor-like domain. / Kringle-like fold / EGF-like domain profile. / EGF-like domain signature 2. / EGF-like domain signature 1. / EGF-like domain / Cupredoxin / Galactose-binding-like domain superfamily / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / 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: 4.1 Å
AuthorsDi Cera, E. / Ruben, E.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Blood / Year: 2022
Title: Cryo-EM structure of the prothrombin-prothrombinase complex.
Authors: Eliza A Ruben / Brock Summers / Michael J Rau / James A J Fitzpatrick / Enrico Di Cera /
Abstract: The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and ...The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.
History
DepositionJan 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 4, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Prothrombin
A: Factor X light chain
C: Coagulation factor Va
D: Coagulation factor Va
B: Activated factor Xa heavy chain


Theoretical massNumber of molelcules
Total (without water)266,2055
Polymers266,2055
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Prothrombin / Thrombin / 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 / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00734, thrombin
#2: Protein Factor X light chain /


Mass: 15743.385 Da / Num. of mol.: 1 / Fragment: UNP residues 41-179
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F10 / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00742
#3: Protein Coagulation factor Va / 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) / References: UniProt: P12259
#4: Protein Coagulation factor Va / 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) / References: UniProt: P12259
#5: Protein Activated factor Xa heavy chain


Mass: 28534.596 Da / Num. of mol.: 1 / Fragment: UNP residues 235-488 / Mutation: S379A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F10 / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P00742

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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
IDNameTypeEntity IDParent-IDSource
1prothrombin prothrombinaseCOMPLEXall0MULTIPLE SOURCES
2Prothrombin (E.C.3.4.21.5), Factor X light chain, Activated factor Xa heavy chainCOMPLEX#1-#2, #51RECOMBINANT
3Coagulation factor VaCOMPLEX#3-#41NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)Organism: Mesocricetus auratus (golden hamster)
Buffer solutionpH: 7.4 / Details: 20mM Hepes, 150mM NaCl, 5mM CaCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 330317 / Symmetry type: POINT

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