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- EMDB-29011: Cryo-EM structure of coagulation factor V short -

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Basic information

Entry
Database: EMDB / ID: EMD-29011
TitleCryo-EM structure of coagulation factor V short
Map datacomposite map used for model building
Sample
  • Tissue: Recombinantly expressed coagulation factor V short full length with a c-terminus HPC4 tag
    • Protein or peptide: Coagulation factor VCoagulation
KeywordsBLOOD CLOTTING
Function / homology
Function and homology information


response to vitamin K / platelet alpha granule / Cargo concentration in the ER / blood circulation / COPII-mediated vesicle transport / COPII-coated ER to Golgi transport vesicle / Common Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / Post-translational protein phosphorylation ...response to vitamin K / platelet alpha granule / Cargo concentration in the ER / blood circulation / COPII-mediated vesicle transport / COPII-coated ER to Golgi transport vesicle / Common Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / extracellular vesicle / blood coagulation / Platelet degranulation / copper ion binding / endoplasmic reticulum lumen / 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. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Multicopper oxidase, N-terminal ...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. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Multicopper oxidase, N-terminal / Multicopper oxidase / Cupredoxin / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Coagulation factor V
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMohammed BM / Pelc LA / Rau MJ / Di Cera E
Funding support United States, 3 items
OrganizationGrant numberCountry
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
CitationJournal: Blood / Year: 2023
Title: Cryo-EM structure of coagulation factor V short.
Authors: Bassem M Mohammed / Leslie A Pelc / Michael J Rau / Enrico Di Cera /
Abstract: Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the ...Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved because of an intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for the binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and revealed the arrangement of the entire A1-A2-B-A3-C1-C2 assembly. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2, and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a potential binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly to the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S, and fXa.
History
DepositionDec 3, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateJul 12, 2023-
Current statusJul 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29011.png

Downloads & links

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Map

FileDownload / File: emd_29011.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map used for model building
Voxel sizeX=Y=Z: 0.9 Å
Density
Contour LevelBy AUTHOR: 2.686
Minimum - Maximum-37.876643999999999 - 65.976425000000006
Average (Standard dev.)-0.000000000002869 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Recombinantly expressed coagulation factor V short full length wi...

EntireName: Recombinantly expressed coagulation factor V short full length with a c-terminus HPC4 tag
Components
  • Tissue: Recombinantly expressed coagulation factor V short full length with a c-terminus HPC4 tag
    • Protein or peptide: Coagulation factor VCoagulation

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Supramolecule #1: Recombinantly expressed coagulation factor V short full length wi...

SupramoleculeName: Recombinantly expressed coagulation factor V short full length with a c-terminus HPC4 tag
type: tissue / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human) / Organ: Liver / Tissue: Blood

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Macromolecule #1: Coagulation factor V

MacromoleculeName: Coagulation factor V / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: Liver / Tissue: Blood
Molecular weightTheoretical: 172.748234 KDa
Recombinant expressionOrganism: Homo sapiens (human)
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 DSYEIFEPPE STVMATRKMH DRLEPEDEES DADYDYQNRL AAALGIRSFR NSSLNQEE E EFNLTALALE NGTEFVSSNT DIIVGSNYSS PSNILGQMPS PSSPTLNDTF LSKEFNPLVI VGLSKDGTDY IEIIPKEEV QSSEDDYAEI DYVPYDDPYK TDVRTNINSS RDPDNIAAWY LRSNNGNRRN YYIAAEEISW DYSEFVQRET DIEDSDDIPE DTTYKKVVF RKYLDSTFTK RDPRGEYEEH LGILGPIIRA EVDDVIQVRF KNLASRPYSL HAHGLSYEKS SEGKTYEDDS P EWFKEDNA VQPNSSYTYV WHATERSGPE SPGSACRAWA YYSAVNPEKD IHSGLIGPLL ICQKGILHKD SNMPVDMREF VL LFMTFDE KKSWYYEKKS RSSWRLTSSE MKKSHEFHAI NGMIYSLPGL KMYEQEWVRL HLLNIGGSQD IHVVHFHGQT LLE NGNKQH QLGVWPLLPG SFKTLEMKAS KPGWWLLNTE VGENQRAGMQ TPFLIMDRDC RMPMGLSTGI ISDSQIKASE FLGY WEPRL ARLNNGGSYN AWSVEKLAAE FASKPWIQVD MQKEVIITGI QTQGAKHYLK SCYTTEFYVA YSSNQINWQI FKGNS TRNV MYFNGNSDAS TIKENQFDPP IVARYIRISP TRAYNRPTLR LELQGCEVNG CSTPLGMENG KIENKQITAS SFKKSW WGD YWEPFRARLN AQGRVNAWQA KANNNKQWLE IDLLKIKKIT AIITQGCKSL SSEMYVKSYT IHYSEQGVEW KPYRLKS SM VDKIFEGNTN TKGHVKNFFN PPIISRFIRV IPKTWNQSIA LRLELFGCDI YEDQVDPRLI DGK

UniProtKB: Coagulation factor V, Coagulation factor V

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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

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMNaClSodium chlorideSodium Chloride
5.0 mMCaCl2Calcium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Details: 2 second blot 20 second wait time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Specialist opticsSpherical aberration corrector: Cs corrector
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-46 / Number grids imaged: 1 / Number real images: 3316 / Average exposure time: 9.36 sec. / Average electron dose: 55.09 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7kve

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V4.0.3) / Number images used: 147000

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8fdg:
Cryo-EM structure of coagulation factor V short

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