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

Basic information

Entry

Database: EMDB / ID: EMD-42405

• Title: Preliminary map of the Prothrombin-prothrombinase complex on nano discs
• Map data: Unsharpened 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

• Keywords: Coagulation / Prothrombin / Prothrombinase / nanodisc / complex / BLOOD CLOTTING

Function / homology

Function:

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

Domain/homology:

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

Component:

Prothrombin / Coagulation factor X / Coagulation factor V

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 6.47 Å
• Authors: Stojanovski BM / Mohammed BM / Di Cera E

Funding support

United States, 8 items

Organization	Grant number	Country
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 Hospital	CDI-CORE-2015-505	United States
Childrens Discovery Institute of Washington University and St. Louis Childrens Hospital	CDI-CORE-2019-813	United States
Foundation for Barnes-Jewish Hospital	3770	United States
Other private	Washington University Diabetes Research Center DK020579
Other private	The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine CA091842

• Citation: Journal: 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 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

Deposition	Oct 19, 2023	-
Header (metadata) release	Nov 1, 2023	-
Map release	Nov 1, 2023	-
Update	Jul 24, 2024	-
Current status	Jul 24, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_42405.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Unsharpened map
• Voxel size: X=Y=Z: 0.94 Å

Density

Contour Level	By AUTHOR: 0.0839
Minimum - Maximum	-0.19871338 - 0.76784295
Average (Standard dev.)	0.0016278322 (±0.028282389)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 512 512 512 Spacing 512 512 512
Cell	A=B=C: 481.28 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_42405_msk_1.map

Additional map: original half-map A

• File: emd_42405_additional_1.map
• Annotation: original half-map A

Additional map: original half-map B

• File: emd_42405_additional_2.map
• Annotation: original half-map B

Half map: half map B filtered to resolution (6.5A)

• File: emd_42405_half_map_1.map
• Annotation: half map B filtered to resolution (6.5A)

Half map: half map A filtered to resolution (6.5A)

• File: emd_42405_half_map_2.map
• Annotation: half map A filtered to resolution (6.5A)

Sample components

Entire : Prothrombin_Prothrombinase complex on lipid nanodiscs.

• Entire: Name: 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

Supramolecule #1: Prothrombin_Prothrombinase complex on lipid nanodiscs.

• Supramolecule: Name: 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). 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

Supramolecule #2: Coagulation Factor Va

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

Supramolecule #3: Coagulation Factor Xa

• Supramolecule: Name: 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

Supramolecule #4: Prothrombin

• Supramolecule: Name: 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

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

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

Electron microscopy

• Microscope: TFS GLACIOS
• Image recording: Film or detector model: FEI FALCON IV (4k x 4k) / Number real images: 2390 / Average electron dose: 51.28 e/Ų
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 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 stage: Cooling holder cryogen: NITROGEN

Image processing

• Startup model: Type of model: NONE
• Final reconstruction: Applied 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 assignment: Type: NOT APPLICABLE
• Final angle assignment: Type: NOT APPLICABLE
• Final 3D classification: Number classes: 3 / Avg.num./class: 3000 / Software - Name: cryoSPARC (ver. 4.3.1)

Atomic model buiding 1

• Refinement: Protocol: AB INITIO MODEL

Output model

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