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- PDB-9p0x: Nanodisc-embedded human TF/FVIIa/XK1 in complex with 10H10 Fab (n... -

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Basic information

Entry
Database: PDB / ID: 9p0x
TitleNanodisc-embedded human TF/FVIIa/XK1 in complex with 10H10 Fab (nanodisc-subtracted)
Components
  • (Human 10H10 antibody Fab ...) x 2
  • Coagulation factor VII Heavy Chain
  • Factor VII light chain
  • Factor X light chain
  • Tissue factor pathway inhibitor
  • Tissue factor,Maltose/maltodextrin-binding periplasmic protein
KeywordsBLOOD CLOTTING / complex / nanodisc
Function / homology
Function and homology information


activation of plasma proteins involved in acute inflammatory response / activation of blood coagulation via clotting cascade / coagulation factor VIIa / response to Thyroid stimulating hormone / response to astaxanthin / response to thyrotropin-releasing hormone / response to 2,3,7,8-tetrachlorodibenzodioxine / response to carbon dioxide / response to genistein / serine-type peptidase complex ...activation of plasma proteins involved in acute inflammatory response / activation of blood coagulation via clotting cascade / coagulation factor VIIa / response to Thyroid stimulating hormone / response to astaxanthin / response to thyrotropin-releasing hormone / response to 2,3,7,8-tetrachlorodibenzodioxine / response to carbon dioxide / response to genistein / serine-type peptidase complex / response to vitamin K / coagulation factor Xa / positive regulation of platelet-derived growth factor receptor signaling pathway / positive regulation of leukocyte chemotaxis / response to thyroxine / NGF-stimulated transcription / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / response to cholesterol / cytokine receptor activity / response to growth hormone / positive regulation of positive chemotaxis / Extrinsic Pathway of Fibrin Clot Formation / endopeptidase inhibitor activity / cellular response to steroid hormone stimulus / positive regulation of endothelial cell apoptotic process / negative regulation of blood coagulation / carbohydrate transmembrane transporter activity / positive regulation of blood coagulation / animal organ regeneration / maltose binding / positive regulation of TOR signaling / maltose transport / maltodextrin transmembrane transport / 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 / side of membrane / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / Intrinsic Pathway of Fibrin Clot Formation / positive regulation of endothelial cell proliferation / serine-type peptidase activity / BMAL1:CLOCK,NPAS2 activates circadian expression / positive regulation of interleukin-8 production / serine-type endopeptidase inhibitor activity / circadian rhythm / phospholipid binding / caveola / protein processing / Golgi lumen / response to estrogen / cytokine-mediated signaling pathway / positive regulation of angiogenesis / blood coagulation / response to estradiol / : / outer membrane-bounded periplasmic space / protease binding / vesicle / response to hypoxia / positive regulation of cell migration / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / external side of plasma membrane / calcium ion binding / positive regulation of gene expression / cell surface / endoplasmic reticulum / proteolysis / extracellular space / extracellular region / membrane / plasma membrane
Similarity search - Function
Tissue factor pathway inhibitor-like / Tissue factor / Tissue factor, conserved site / Tissue factor signature. / Interferon/interleukin receptor domain / Interferon-alpha/beta receptor, fibronectin type III / : / Tissue factor / : / Peptidase S1A, coagulation factor VII/IX/X/C/Z ...Tissue factor pathway inhibitor-like / Tissue factor / Tissue factor, conserved site / Tissue factor signature. / Interferon/interleukin receptor domain / Interferon-alpha/beta receptor, fibronectin type III / : / Tissue factor / : / Peptidase S1A, coagulation factor VII/IX/X/C/Z / : / Coagulation factor-like, Gla domain superfamily / Proteinase inhibitor I2, Kunitz, conserved site / Pancreatic trypsin inhibitor (Kunitz) family signature. / BPTI/Kunitz family of serine protease inhibitors. / Pancreatic trypsin inhibitor Kunitz domain / Kunitz/Bovine pancreatic trypsin inhibitor domain / Pancreatic trypsin inhibitor (Kunitz) family profile. / Pancreatic trypsin inhibitor Kunitz domain superfamily / Coagulation Factor Xa inhibitory site / EGF-like domain / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 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. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / Fibronectin type III / Fibronectin type III 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 / Immunoglobulin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
beta-D-glucopyranose / alpha-L-fucopyranose / Coagulation factor X / Coagulation factor VII / Maltose/maltodextrin-binding periplasmic protein / Tissue factor pathway inhibitor / Tissue factor
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPhotenhauer, A.L. / Sedzro, J.C. / Ohi, M.D. / Morrissey, J.H.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R35 HL135823 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R35 HL171334 United States
National Institutes of Health/Office of the DirectorS10OD030275 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R24 GM145965 United States
American Heart AssociationN028347 United States
CitationJournal: Blood / Year: 2025
Title: Cryo-EM structure of the tissue factor/factor VIIa complex with a factor X mimetic reveals a novel allosteric mechanism.
Authors: Josepha C Sedzro / Amanda L Photenhauer / Fabienne Birkle / Katarina Meze / Alex Mortenson / Cade Duckworth / Po-Chao Wen / Sarah Kearns / Michael A Cianfrocco / Emad Tajkhorshid / Melanie D ...Authors: Josepha C Sedzro / Amanda L Photenhauer / Fabienne Birkle / Katarina Meze / Alex Mortenson / Cade Duckworth / Po-Chao Wen / Sarah Kearns / Michael A Cianfrocco / Emad Tajkhorshid / Melanie D Ohi / James H Morrissey /
Abstract: Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on ...Blood clotting is triggered in hemostasis and thrombosis when the membrane-bound tissue factor (TF)/factor VIIa (FVIIa) complex activates factor X (FX). There are no structures of TF/FVIIa on membranes, with or without FX. Using cryo-EM to address this gap, we assembled TF/FVIIa complexes on nanoscale membrane bilayers (nanodiscs), bound to XK1 and an antibody fragment. XK1 is a FX mimetic whose protease domain is replaced by the first Kunitz-type (K1) domain of tissue factor pathway inhibitor, while 10H10 is a non-inhibitory, anti-TF antibody. We determined a cryo-EM structure of a TF/FVIIa/XK1/10H10/nanodisc complex with a resolution of 3.7 Å, allowing us to model all the protein backbones. TF/FVIIa extends perpendicularly from the membrane, interacting with a "handle shaped" XK1 at two locations: the K1 domain docks into FVIIa's active site, while the γ-carboxyglutamate-rich (GLA) domain binds to TF's substrate-binding exosite. The FX and FVIIa GLA domains also contact each other and the membrane surface. Except for a minor contact between the first epidermal growth factor (EGF)-like domain of XK1 and TF, the rest of the FX light chain does not interact with TF/FVIIa. The structure reveals a previously unrecognized, membrane-dependent allosteric activation mechanism between FVIIa and TF where a serine-rich loop in TF that partially obscures the TF exosite must undergo a shift to allow access of the FX GLA domain to its final binding location on the membrane-bound TF/FVIIa complex. This mechanism also provides a novel explanation for the otherwise puzzling phenomenon of TF encryption/decryption on cell surfaces.
History
DepositionJun 7, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 27, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
V: Factor VII light chain
X: Factor X light chain
S: Coagulation factor VII Heavy Chain
T: Tissue factor,Maltose/maltodextrin-binding periplasmic protein
K: Tissue factor pathway inhibitor
H: Human 10H10 antibody Fab heavy chain
L: Human 10H10 antibody Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)184,26925
Polymers183,3467
Non-polymers92218
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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Protein , 5 types, 5 molecules VXSTK

#1: Protein Factor VII light chain


Mass: 16359.772 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F7 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P08709
#2: Protein Factor X light chain


Mass: 15309.423 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F10 / Plasmid: pcDNA3.1(+) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P00742
#3: Protein Coagulation factor VII Heavy Chain / Proconvertin / Serum prothrombin conversion accelerator / SPCA


Mass: 28103.256 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F7 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P08709, coagulation factor VIIa
#4: Protein Tissue factor,Maltose/maltodextrin-binding periplasmic protein / TF / Coagulation factor III / Thromboplastin / MMBP / Maltodextrin-binding protein / Maltose- ...TF / Coagulation factor III / Thromboplastin / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 67828.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: F3, malE, Z5632, ECs5017 / Plasmid: pET-26b(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P13726, UniProt: P0AEY0
#5: Protein Tissue factor pathway inhibitor / TFPI / Extrinsic pathway inhibitor / EPI / Lipoprotein-associated coagulation inhibitor / LACI


Mass: 7979.198 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TFPI, LACI, TFPI1 / Plasmid: pcDNA3.1(+) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P10646

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Antibody , 2 types, 2 molecules HL

#6: Antibody Human 10H10 antibody Fab heavy chain


Mass: 23569.252 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: BALB/c
#7: Antibody Human 10H10 antibody Fab light chain


Mass: 24196.709 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: BALB/c

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Sugars , 2 types, 2 molecules

#10: Sugar ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DGlcpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-glucopyranoseCOMMON NAMEGMML 1.0
b-D-GlcpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#11: Sugar ChemComp-FUC / alpha-L-fucopyranose / alpha-L-fucose / 6-deoxy-alpha-L-galactopyranose / L-fucose / fucose


Type: L-saccharide, alpha linking / Mass: 164.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O5
IdentifierTypeProgram
LFucpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-L-fucopyranoseCOMMON NAMEGMML 1.0
a-L-FucpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
FucSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 16 molecules

#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#9: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestN
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 factor VIIa, XK1 and the Fab fragment of antibody 10H10, all bound to tissue factor embedded in a nanodisc membrane bilayer.COMPLEXMap with nanodisc subtracted.#1-#70MULTIPLE SOURCES
2Human 10H10 antibody FabCOMPLEX#6-#71NATURAL
3XK1 chimeric proteinCOMPLEX#2, #51RECOMBINANT
4Human factor VIIa (FVIIa)COMPLEX#1, #31RECOMBINANT
5Human MBP-tagged tissue factorCOMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Homo sapiens (human)9606
44Homo sapiens (human)9606
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
33Homo sapiens (human)9606
44Homo sapiens (human)9606HEK-293
55Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7132
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2862371
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20107 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation
Details: Crystal structures were rigid-body fit into the density map and model optimization was then carried out with Phenix real-space refine. Manual refinement in Coot was performed to ensure that ...Details: Crystal structures were rigid-body fit into the density map and model optimization was then carried out with Phenix real-space refine. Manual refinement in Coot was performed to ensure that the backbone traces followed the density in regions where the map was high enough resolution to trace the backbone. Secondary structure restraints were used to ensure that alpha-helices and beta-sheets did not deviate far from their expected geometry. A final check of MolProbity and cross correlation was done to ensure model quality.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue range
11dan

1dan

L1danL46-142146-142
21dan

1dan

H1danH16-257116-257
31dan

1dan

T1danT6-8016-80
41dan

1dan

U1danU91-210191-210
53th2

3th2

L3th2L1-461-46
61iod

1iod

G1iodG401-444401-444
71xka

1xka

L1xkaL49-13949-139
84bqd

4bqd

A4bqdA12-7912-79
91200000000H1200000000H112-214112-214
101200000000L1200000000L107-214107-214
114m7l

4m7l

H4m7lH1-1191-119
124m7l

4m7l

L4m7lL1-1131-113
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 95.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319929
ELECTRON MICROSCOPYf_angle_d0.694913495
ELECTRON MICROSCOPYf_chiral_restr0.04341472
ELECTRON MICROSCOPYf_plane_restr0.00441758
ELECTRON MICROSCOPYf_dihedral_angle_d9.75171434

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