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- EMDB-52585: A 3.3 angstrom cryo-EM structure of an engineered high-affinity h... -

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Entry
Database: EMDB / ID: EMD-52585
TitleA 3.3 angstrom cryo-EM structure of an engineered high-affinity human prothrombinase complex
Map dataunsharpened map
Sample
  • Complex: prothrombinase, a complex of fVa and fXa
    • Protein or peptide: Activated factor Xa heavy chain
    • Protein or peptide: Factor X light chain
    • Protein or peptide: Coagulation factor V heavy chain
    • Protein or peptide: Coagulation factor V light chain
  • Ligand: CALCIUM ION
  • Ligand: SODIUM ION
  • Ligand: L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide
  • Ligand: COPPER (II) ION
Keywordsenzyme / complex / prothrombinase / BLOOD CLOTTING
Function / homology
Function and homology information


response to vitamin K / coagulation factor Xa / platelet alpha granule / Cargo concentration in the ER / COPII-coated ER to Golgi transport vesicle / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / Extrinsic Pathway of Fibrin Clot Formation ...response to vitamin K / coagulation factor Xa / platelet alpha granule / Cargo concentration in the ER / COPII-coated ER to Golgi transport vesicle / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / Extrinsic Pathway of Fibrin Clot Formation / blood circulation / positive regulation of TOR signaling / 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 / Post-translational protein phosphorylation / phospholipid binding / 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 / positive regulation of cell migration / endoplasmic reticulum lumen / copper ion binding / external side of plasma membrane / serine-type endopeptidase activity / calcium ion binding / 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 / : / F5/8 type C domain / Coagulation factor-like, Gla domain superfamily / Coagulation factor 5/8 C-terminal domain / 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 / 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
Coagulation factor X / Coagulation factor V
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsHuntington JA / Faille A / Ustok FI
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
British Heart FoundationRG/16/9/32391 United Kingdom
British Heart FoundationPG/24/11721 United Kingdom
CitationJournal: Blood / Year: 2025
Title: A 3.3-Å cryo-EM structure of an engineered high-affinity human prothrombinase complex.
Authors: Fatma Işık Üstok / Alexandre Faille / James A Huntington /
Abstract: Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor (f) Xa and fVa. The affinity of fXa for fVa is low, with assembly and function ...Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor (f) Xa and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop C-terminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2-peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel b-strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research, and provides important new insights into the assembly and function of the prothrombinase complex.
History
DepositionJan 20, 2025-
Header (metadata) releaseNov 26, 2025-
Map releaseNov 26, 2025-
UpdateNov 26, 2025-
Current statusNov 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52585.png

Downloads & links

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Map

FileDownload / File: emd_52585.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0252
Minimum - Maximum-0.21637994 - 0.45701113
Average (Standard dev.)0.00013841107 (±0.007930974)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 365.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map

Fileemd_52585_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_52585_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_52585_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : prothrombinase, a complex of fVa and fXa

EntireName: prothrombinase, a complex of fVa and fXa
Components
  • Complex: prothrombinase, a complex of fVa and fXa
    • Protein or peptide: Activated factor Xa heavy chain
    • Protein or peptide: Factor X light chain
    • Protein or peptide: Coagulation factor V heavy chain
    • Protein or peptide: Coagulation factor V light chain
  • Ligand: CALCIUM ION
  • Ligand: SODIUM ION
  • Ligand: L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide
  • Ligand: COPPER (II) ION

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Supramolecule #1: prothrombinase, a complex of fVa and fXa

SupramoleculeName: prothrombinase, a complex of fVa and fXa / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: M17-prothrombinase was formed by mixing fVa with EGRCK-inhibited M17-fXa at a 1:6 molar ratio with a final concentration of 1.68uM in 20mM HEPES pH 7.5, 150mM NaCl and 5mM CaCl2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Activated factor Xa heavy chain

MacromoleculeName: Activated factor Xa heavy chain / type: protein_or_peptide / ID: 1
Details: Mutations given using chymotrypsin template numbering as in the coordinates.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.789916 KDa
Recombinant expressionOrganism: Cytomegalovirus
SequenceString: IVGGQECKDG ECPWQALLIN EENEGFCGGT ILSEFYILTA AHCLYQAKRF KVRVGDRNTE QEEGGEAVHE VEVVIKHNRF TKETYDFDI AVLRLKTPIT FRMNVAPACL PERDWANETL MKQDTGIVSG FGRTHEKGRQ STRLKMLEVP YVDRHSCMLS S DFRITQNM ...String:
IVGGQECKDG ECPWQALLIN EENEGFCGGT ILSEFYILTA AHCLYQAKRF KVRVGDRNTE QEEGGEAVHE VEVVIKHNRF TKETYDFDI AVLRLKTPIT FRMNVAPACL PERDWANETL MKQDTGIVSG FGRTHEKGRQ STRLKMLEVP YVDRHSCMLS S DFRITQNM FCAGYDTKQE DACQGDSGGP HVTRFKDTYF VTGIVSWGEG CARKGKYGIY TKVTRFLKWI KRSMKTRGLP KA KSHAPEV ITSSPLK

UniProtKB: Coagulation factor X

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Macromolecule #2: Factor X light chain

MacromoleculeName: Factor X light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.765613 KDa
Recombinant expressionOrganism: Cytomegalovirus
SequenceString:
ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN CELFTRKLC RAFNGDCDQF CKRVQSSVVC SCARGYTLAD NGKACIPTGP YPCGKQTLER

UniProtKB: Coagulation factor X

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Macromolecule #3: Coagulation factor V heavy chain

MacromoleculeName: Coagulation factor V heavy chain / type: protein_or_peptide / ID: 3 / Details: TYS is a sulphated Tyrosine. / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 81.354453 KDa
Recombinant expressionOrganism: unidentified plasmid (others)
SequenceString: AQLRQFYVAA QGISWSYRPE PTNSSLNLSV TSFKKIVYRE YEPYFKKEKP QSTISGLLGP TLYAEVGDII KVHFKNKADK PLSIHPQGI RYSKLSEGAS YLDHTFPAEK MDDAVAPGRE YTYEWSISED SGPTHDDPPC LTHIYYSHEN LIEDFNSGLI G PLLICKKG ...String:
AQLRQFYVAA QGISWSYRPE PTNSSLNLSV TSFKKIVYRE YEPYFKKEKP QSTISGLLGP TLYAEVGDII KVHFKNKADK PLSIHPQGI RYSKLSEGAS YLDHTFPAEK MDDAVAPGRE YTYEWSISED SGPTHDDPPC LTHIYYSHEN LIEDFNSGLI G PLLICKKG TLTEGGTQKT FDKQIVLLFA VFDESKSWSQ SSSLMYTVNG YVNGTMPDIT VCAHDHISWH LLGMSSGPEL FS IHFNGQV LEQNHHKVSA ITLVSATSTT ANMTVGPEGK WIISSLTPKH LQAGMQAYID IKNCPKKTRN LKKITREQRR HMK RWEYFI AAEEVIWDYA PVIPANMDKK YRSQHLDNFS NQIGKHYKKV MYTQYEDESF TKHTVNPNMK EDGILGPIIR AQVR DTLKI VFKNMASRPY SIYPHGVTFS PYEDEVNSSF TSGRNNTMIR AVQPGETYTY KWNILEFDEP TENDAQCLTR PYYSD VDIM RDIASGLIGL LLICKSRSLD RRGIQRAADI EQQAVFAVFD ENKSWYLEDN INKFCENPDE VKRDDPKFYE SNIMST ING YVPESITTLG FCFDDTVQWH FCSVGTQNEI LTIHFTGHSF IYGKRHEDTL TLFPMRGESV TVTMDNVGTW MLTSMNS SP RSKKLRLKFR DVKCIPDDDE DS(TYS)EIFEPPE STVMATRKMH DRLEPEDEES DADYDYQNRL AAALGIR

UniProtKB: Coagulation factor V

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Macromolecule #4: Coagulation factor V light chain

MacromoleculeName: Coagulation factor V light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 75.283008 KDa
Recombinant expressionOrganism: unidentified plasmid (others)
SequenceString: SNNGNRRNYY IAAEEISWDY SEFVQRETDI EDSDDIPEDT TYKKVVFRKY LDSTFTKRDP RGEYEEHLGI LGPIIRAEVD DVIQVRFKN LASRPYSLHA HGLSYEKSSE GKTYEDDSPE WFKEDNAVQP NSSYTYVWHA TERSGPESPG SACRAWAYYS A VNPEKDIH ...String:
SNNGNRRNYY IAAEEISWDY SEFVQRETDI EDSDDIPEDT TYKKVVFRKY LDSTFTKRDP RGEYEEHLGI LGPIIRAEVD DVIQVRFKN LASRPYSLHA HGLSYEKSSE GKTYEDDSPE WFKEDNAVQP NSSYTYVWHA TERSGPESPG SACRAWAYYS A VNPEKDIH SGLIGPLLIC QKGILHKDSN MPMDMREFVL LFMTFDEKKS WYYEKKSRSS WRLTSSEMKK SHEFHAINGM IY SLPGLKM YEQEWVRLHL LNIGGSQDIH VVHFHGQTLL ENGNKQHQLG VWPLLPGSFK TLEMKASKPG WWLLNTEVGE NQR AGMQTP FLIMDRDCRM PMGLSTGIIS DSQIKASEFL GYWEPRLARL NNGGSYNAWS VEKLAAEFAS KPWIQVDMQK EVII TGIQT QGAKHYLKSC YTTEFYVAYS SNQINWQIFK GNSTRNVMYF NGNSDASTIK ENQFDPPIVA RYIRISPTRA YNRPT LRLE LQGCEVNGCS TPLGMENGKI ENKQITASSF KKSWWGDYWE PFRARLNAQG RVNAWQAKAN NNKQWLEIDL LKIKKI TAI ITQGCKSLSS EMYVKSYTIH YSEQGVEWKP YRLKSSMVDK IFEGNTNTKG HVKNFFNPPI ISRFIRVIPK TWNQSIA LR LELFGCDIY

UniProtKB: Coagulation factor V

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Macromolecule #9: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #10: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 10 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #11: L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2...

MacromoleculeName: L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide
type: ligand / ID: 11 / Number of copies: 1 / Formula: 0GJ
Molecular weightTheoretical: 395.862 Da
Chemical component information

ChemComp-0GJ:
L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide

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Macromolecule #12: COPPER (II) ION

MacromoleculeName: COPPER (II) ION / type: ligand / ID: 12 / Number of copies: 1 / Formula: CU
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU:
COPPER (II) ION

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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: 7.5 / Details: 20mM HEPES, 150mM NaCl and 5mM CaCl2
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa / Details: using a Pelco EasiGlow
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsM17-prothrombinase was formed by mixing fVa with EGRCK-inhibited M17-fXa at a 1:6 molar ratio with a final concentration of 1.68uM

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.74 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70154
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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