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- EMDB-29009: Cryo-EM structure of coagulation factor V short -

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Basic information

Entry
Database: EMDB / ID: EMD-29009
TitleCryo-EM structure of coagulation factor V short
Map dataPrimary Map
Sample
  • Tissue: Recombinantly expressed coagulation factor V short full length with a c-terminus HPC4 tag
    • Protein or peptide: Coagulation Factor V short
KeywordsBLOOD CLOTTING
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-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29009.png

Downloads & links

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Map

FileDownload / File: emd_29009.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary Map
Voxel sizeX=Y=Z: 0.9 Å
Density
Contour LevelBy AUTHOR: 0.0958
Minimum - Maximum-1.206976 - 1.8699639
Average (Standard dev.)0.00039385832 (±0.030459873)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_29009_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_29009_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_29009_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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 V short

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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 short

MacromoleculeName: Coagulation Factor V short / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: Liver / Tissue: Blood
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AQLRQFYVAA QGISWSYRPE PTNSSLNLSV TSFKKIVYRE YEPYFKKEKP QSTISGLLGP TLYAEVGDII KVHFKNKADK PLSIHPQGIR YSKLSEGASY LDHTFPAEKM DDAVAPGREY TYEWSISEDS GPTHDDPPCL THIYYSHENL IEDFNSGLIG PLLICKKGTL ...String:
AQLRQFYVAA QGISWSYRPE PTNSSLNLSV TSFKKIVYRE YEPYFKKEKP QSTISGLLGP TLYAEVGDII KVHFKNKADK PLSIHPQGIR YSKLSEGASY LDHTFPAEKM DDAVAPGREY TYEWSISEDS GPTHDDPPCL THIYYSHENL IEDFNSGLIG PLLICKKGTL TEGGTQKTFD KQIVLLFAVF DESKSWSQSS SLMYTVNGYV NGTMPDITVC AHDHISWHLL GMSSGPELFS IHFNGQVLEQ NHHKVSAITL VSATSTTANM TVGPEGKWII SSLTPKHLQA GMQAYIDIKN CPKKTRNLKK ITREQRRHMK RWEYFIAAEE VIWDYAPVIP ANMDKKYRSQ HLDNFSNQIG KHYKKVMYTQ YEDESFTKHT VNPNMKEDGI LGPIIRAQVR DTLKIVFKNM ASRPYSIYPH GVTFSPYEDE VNSSFTSGRN NTMIRAVQPG ETYTYKWNIL EFDEPTENDA QCLTRPYYSD VDIMRDIASG LIGLLLICKS RSLDRRGIQR AADIEQQAVF AVFDENKSWY LEDNINKFCE NPDEVKRDDP KFYESNIMST INGYVPESIT TLGFCFDDTV QWHFCSVGTQ NEILTIHFTG HSFIYGKRHE DTLTLFPMRG ESVTVTMDNV GTWMLTSMNS SPRSKKLRLK FRDVKCIPDD DEDSYEIFEP PESTVMATRK MHDRLEPEDE ESDADYDYQN RLAAALGIRS FRNSSLNQEE EEFNLTALAL ENGTEFVSSN TDIIVGSNYS SPSNILGQMP SPSSPTLNDT FLSKEFNPLV IVGLSKDGTD YIEIIPKEEV QSSEDDYAEI DYVPYDDPYK TDVRTNINSS RDPDNIAAWY LRSNNGNRRN YYIAAEEISW DYSEFVQRET DIEDSDDIPE DTTYKKVVFR KYLDSTFTKR DPRGEYEEHL GILGPIIRAE VDDVIQVRFK NLASRPYSLH AHGLSYEKSS EGKTYEDDSP EWFKEDNAVQ PNSSYTYVWH ATERSGPESP GSACRAWAYY SAVNPEKDIH SGLIGPLLIC QKGILHKDSN MPVDMREFVL LFMTFDEKKS WYYEKKSRSS WRLTSSEMKK SHEFHAINGM IYSLPGLKMY EQEWVRLHLL NIGGSQDIHV VHFHGQTLLE NGNKQHQLGV WPLLPGSFKT LEMKASKPGW WLLNTEVGEN QRAGMQTPFL IMDRDCRMPM GLSTGIISDS QIKASEFLGY WEPRLARLNN GGSYNAWSVE KLAAEFASKP WIQVDMQKEV IITGIQTQGA KHYLKSCYTT EFYVAYSSNQ INWQIFKGNS TRNVMYFNGN SDASTIKENQ FDPPIVARYI RISPTRAYNR PTLRLELQGC EVNGCSTPLG MENGKIENKQ ITASSFKKSW WGDYWEPFRA RLNAQGRVNA WQAKANNNKQ WLEIDLLKIK KITAIITQGC KSLSSEMYVK SYTIHYSEQG VEWKPYRLKS SMVDKIFEGN TNTKGHVKNF FNPPIISRFI RVIPKTWNQS ITLRLELFGC DIYEDQVDPR LIDGK

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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
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
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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