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- EMDB-71093: Nanodisc-embedded human TF/FVIIa/XK1 in complex with 10H10 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-71093
TitleNanodisc-embedded human TF/FVIIa/XK1 in complex with 10H10 Fab
Map dataUnsharpened cryoem map of TF/FVIIa/XK1 complex in nanodiscs without particle subtraction
Sample
  • Complex: Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H10, all bound to tissue factor embedded in a nanodisc membrane bilayer.
    • Complex: Human 10H10 antibody Fab
      • Protein or peptide: Human 10H10 antibody Fab heavy chain
      • Protein or peptide: Human 10H10 antibody Fab light chain
    • Complex: XK1 chimeric protein
      • Protein or peptide: Human factor X (FX) light chain
      • Protein or peptide: Human TFPI Kunitz domain 1 (K1)
    • Complex: Human activated factor VIIa
      • Protein or peptide: Human factor VIIa (FVIIa) light chain
      • Protein or peptide: Human factor VIIa (FVIIa) protease domain
    • Complex: Human MBP-tagged tissue factor
      • Protein or peptide: Human tissue factor (TF)
Keywordscomplex / nanodisc / blood clotting
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPhotenhauer AL / Sedzro JC / Morrissey JH / Ohi MD
Funding support United States, 5 items
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 9, 2025-
Header (metadata) releaseAug 27, 2025-
Map releaseAug 27, 2025-
UpdateAug 27, 2025-
Current statusAug 27, 2025Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_71093.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened cryoem map of TF/FVIIa/XK1 complex in nanodiscs without particle subtraction
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.3066422 - 0.61088324
Average (Standard dev.)-0.0000669639 (±0.010212363)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 350.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H1...

EntireName: Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H10, all bound to tissue factor embedded in a nanodisc membrane bilayer.
Components
  • Complex: Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H10, all bound to tissue factor embedded in a nanodisc membrane bilayer.
    • Complex: Human 10H10 antibody Fab
      • Protein or peptide: Human 10H10 antibody Fab heavy chain
      • Protein or peptide: Human 10H10 antibody Fab light chain
    • Complex: XK1 chimeric protein
      • Protein or peptide: Human factor X (FX) light chain
      • Protein or peptide: Human TFPI Kunitz domain 1 (K1)
    • Complex: Human activated factor VIIa
      • Protein or peptide: Human factor VIIa (FVIIa) light chain
      • Protein or peptide: Human factor VIIa (FVIIa) protease domain
    • Complex: Human MBP-tagged tissue factor
      • Protein or peptide: Human tissue factor (TF)

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Supramolecule #1: Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H1...

SupramoleculeName: Complex of factor VIIa, XK1 and the Fab fragment of antibody 10H10, all bound to tissue factor embedded in a nanodisc membrane bilayer.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Map with nanodisc.

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Supramolecule #2: Human 10H10 antibody Fab

SupramoleculeName: Human 10H10 antibody Fab / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #6-#7
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: XK1 chimeric protein

SupramoleculeName: XK1 chimeric protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Human activated factor VIIa

SupramoleculeName: Human activated factor VIIa / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: Human MBP-tagged tissue factor

SupramoleculeName: Human MBP-tagged tissue factor / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Human factor VIIa (FVIIa) light chain

MacromoleculeName: Human factor VIIa (FVIIa) light chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ANAFL(CGU)(CGU)LRP GSL(CGU)R(CGU)CK(CGU)(CGU) QCSF(CGU)(CGU)AR(CGU)I FKDA(CGU)RTKLF

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Macromolecule #2: Human factor VIIa (FVIIa) protease domain

MacromoleculeName: Human factor VIIa (FVIIa) protease domain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
IVGGKVCPKG ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR NLIAVLGEHD LSEHDGDEQS RRVAQVIIPS

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Macromolecule #3: Human factor X (FX) light chain

MacromoleculeName: Human factor X (FX) light chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ANSFL(CGU)(CGU)MKK GHL(CGU)R(CGU)CM(CGU)(CGU) TCSY(CGU)(CGU)AR(CGU)V F(CGU)DSDKTN

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Macromolecule #4: Human TFPI Kunitz domain 1 (K1)

MacromoleculeName: Human TFPI Kunitz domain 1 (K1) / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
TDTELPPLKL MHSFCAFKAD DGPCKAIMKR FFFNIFTRQC EEFIYGGCEG NQNRFESLEE CKKMCTRD

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Macromolecule #5: Human tissue factor (TF)

MacromoleculeName: Human tissue factor (TF) / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
TVAAYNLTWK STNFKTILEW EPKPVNQVYT VQISTKSGDW KSKCFYTTDT ECDLTDEIVK DVKQTYLARV FSYPAGNVES

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Macromolecule #6: Human 10H10 antibody Fab heavy chain

MacromoleculeName: Human 10H10 antibody Fab heavy chain / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
QVHLQQSGAE LMKPGASVKI SCKASGYTFI TYWIEWVKQR PGHGLEWIGD ILPGSGSTNY NENFKGKATF TADSSSNTAY

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Macromolecule #7: Human 10H10 antibody Fab light chain

MacromoleculeName: Human 10H10 antibody Fab light chain / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL SSGNQKNYLT WYQQIPGQPP KLLIYWASTR ESGVPDRFTG SGSGTDFTLT

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7132 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3440922
CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Software - details: Non-uniform refinement / Number images used: 91001
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

9p0x


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Model generated from associated entry

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