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- PDB-9mot: Cryo-EM structure of factor Va bound to activated protein C -

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Basic information

Entry
Database: PDB / ID: 9mot
TitleCryo-EM structure of factor Va bound to activated protein C
Components
  • Coagulation factor Va heavy chain
  • Coagulation factor Va light chain
  • Vitamin K-dependent protein C heavy chain
KeywordsBLOOD CLOTTING / Coagulation / Activated Factor V / Activated Protein C
Function / homology
Function and homology information


activated protein C (thrombin-activated peptidase) / positive regulation of establishment of endothelial barrier / negative regulation of coagulation / response to vitamin K / platelet alpha granule / Cargo concentration in the ER / COPII-mediated vesicle transport / blood circulation / COPII-coated ER to Golgi transport vesicle / negative regulation of blood coagulation ...activated protein C (thrombin-activated peptidase) / positive regulation of establishment of endothelial barrier / negative regulation of coagulation / response to vitamin K / platelet alpha granule / Cargo concentration in the ER / COPII-mediated vesicle transport / blood circulation / COPII-coated ER to Golgi transport vesicle / negative regulation of blood coagulation / 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 / Intrinsic Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / Cell surface interactions at the vascular wall / Post-translational protein phosphorylation / negative regulation of inflammatory response / Golgi lumen / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / blood coagulation / Platelet degranulation / extracellular vesicle / copper ion binding / endoplasmic reticulum lumen / serine-type endopeptidase activity / calcium ion binding / negative regulation of apoptotic process / endoplasmic reticulum / Golgi apparatus / proteolysis / extracellular space / 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 / Coagulation Factor Xa inhibitory site / Multicopper oxidase, N-terminal / Multicopper oxidase / 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 / 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. / 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
Vitamin K-dependent protein C / Coagulation factor V
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMohammed, B.M. / Basore, K. / Di Cera, E.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL049413, HL139554 and HL147821 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalCDI-CORE-2015-505 and CDI-CORE-2019-813 United States
The Foundation for Barnes-Jewish Hospital3770 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK020579 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA091842 United States
CitationJournal: Blood / Year: 2025
Title: Cryo-EM structure of coagulation factor Va bound to activated protein C.
Authors: Bassem M Mohammed / Katherine Basore / Enrico Di Cera /
Abstract: Coagulation factor Va (fVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In ...Coagulation factor Va (fVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, fVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the fVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of fVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here we report the cryo-EM structure of the fVa-APC complex at 3.15 Å resolution where the protease domain of APC engages R506 in the A2 domain of fVa mainly through electrostatic interactions between positively charged residues in the 30- and 70- loops of APC and an electronegative surface of fVa. The auxiliary Gla and EGF domains of APC are highly dynamic and point to solvent, without making contacts with fVa. Binding of APC displaces a large portion of the A2 domain of fVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch", or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the fVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.
History
DepositionDec 27, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Coagulation factor Va heavy chain
B: Coagulation factor Va light chain
D: Vitamin K-dependent protein C heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)185,27711
Polymers183,5073
Non-polymers1,7708
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
DetailsA complex of coagulation factor Va (Chain A and Chain B) and Activated protein C (Chain D)

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Components

#1: Protein 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 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
#3: Protein Vitamin K-dependent protein C heavy chain


Mass: 26949.836 Da / Num. of mol.: 1 / Mutation: S360A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Tissue: Blood / Gene: PROC / Plasmid: pRC-RSV / Cell line (production host): BHK / Production host: Mesocricetus auratus (golden hamster) / References: UniProt: P04070
#4: 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.: 8 / Source method: isolated from a natural source / 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
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of coagulation Factor Va and Activated Protein CCOMPLEXCoagulation activated Factor V (FVa) [from plasma] complex with Activated Protein C (APC) [made recombinantly with Ser360Ala mutation].#1-#30MULTIPLE SOURCES
2Coagulation Factor VaCOMPLEX#1-#21NATURAL
3Activated Protein CCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDTissue
22Homo sapiens (human)9606Blood
33Homo sapiens (human)9606
Source (recombinant)Organism: Mesocricetus auratus (golden hamster)
Buffer solution
IDSpecimen-IDpHDetails
117.420 mM HEPES, 150 mM NaCl, 5 mM CaCl2
227.420 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 40uM n-Dodecyl-B-D-Maltoside
337.420 mM HEPES, 150 mM NaCl, 5 mM CaCl2
Specimen

Experiment-ID: 1 / Conc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES

IDDetails
10.1 mg/mL
2
3
Specimen support
IDSpecimen-IDDetailsGrid materialGrid mesh size (divisions/in.)Grid type
11Grids were coated with polylysineCOPPER300Quantifoil R1.2/1.3
22COPPER300Quantifoil R1.2/1.3
33COPPER300Quantifoil R1.2/1.3
Vitrification
IDInstrumentCryogen nameHumidity (%)Specimen-IDChamber temperature (K)Entry-ID
1FEI VITROBOT MARK IVETHANE1001277.159MOT
2FEI VITROBOT MARK IVETHANE1002277.159MOT
3FEI VITROBOT MARK IVETHANE1003277.159MOT

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1

IDElectron dose (e/Å2)Detector modeNum. of real imagesDetails
151.86COUNTING319330 degrees tilt
246.6600
346.89265530 degrees tilt
447.192379
546.89420
652.83210
Image scans
WidthHeightMovie frames/imageIDImage recording-IDEntry-ID
4096409650119MOT
40964096229MOT
40964096339MOT
40964096449MOT
40964096559MOT
40964096669MOT

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.2particle selection
2EPUimage acquisition
4cryoSPARC4.6.2CTF correction
7UCSF ChimeraX1.9model fitting
12cryoSPARC4.6.23D reconstruction
13PHENIX1.21.2-5419model refinement
14Coot0.9.8.95model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 384239 / Algorithm: BACK PROJECTION
Details: 3D flex with custom mesh was used for the reconstruction
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18TN9

8tn9

18TN91PDBexperimental model
29CTH

9cth

19CTH2PDBexperimental model
31AUT

1aut

11AUT3PDBexperimental model

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