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- EMDB-29770: Coagulation factor VIII bound to a patient-derived anti-C1 domain... -

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Basic information

Entry
Database: EMDB / ID: EMD-29770
TitleCoagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
Map dataBlood coagulation factor VIII bound to patient-derived anti-C1 domain antibody inhibitor
Sample
  • Complex: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
    • Protein or peptide: Coagulation factor VIII chimera from human and pig
    • Protein or peptide: NB33 light chain
    • Protein or peptide: NB33 heavy chain
KeywordsImmune system / BLOOD CLOTTING
Function / homology
Function and homology information


Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 sulfation at Y1699 / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant ...Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 sulfation at Y1699 / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / COPII-coated ER to Golgi transport vesicle / Defective F8 cleavage by thrombin / Common Pathway of Fibrin Clot Formation / Intrinsic Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / acute-phase response / Golgi lumen / blood coagulation / Platelet degranulation / signaling receptor activity / oxidoreductase activity / copper ion binding / endoplasmic reticulum lumen / extracellular space / extracellular region / 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. / Multicopper oxidase, C-terminal / Multicopper oxidase / F5/8 type C domain ...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. / Multicopper oxidase, C-terminal / Multicopper oxidase / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Multicopper oxidase, type 1 / Multicopper oxidase / Multicopper oxidase, N-terminal / Multicopper oxidase / Cupredoxin / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Coagulation factor VIII / Coagulation factor VIII
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.23 Å
AuthorsChilders KC / Davulcu O / Haynes RM / Lollar P / Doering CB / Coxon CH / Spiegel PC
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R15HL135658 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)U54HL141981 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R44HL117511 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R44HL110448 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Blood / Year: 2023
Title: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor.
Authors: Kenneth C Childers / Nathan G Avery / Kevin A Estrada Alamo / Omar Davulcu / Rose Marie Haynes / Pete Lollar / Christopher B Doering / Carmen H Coxon / P Clint Spiegel /
Abstract: The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in ...The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1-mediated clearance.
History
DepositionFeb 15, 2023-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29770.png

Downloads & links

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Map

FileDownload / File: emd_29770.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBlood coagulation factor VIII bound to patient-derived anti-C1 domain antibody inhibitor
Voxel sizeX=Y=Z: 0.788 Å
Density
Contour LevelBy AUTHOR: 0.0128
Minimum - Maximum-0.10766629 - 0.19333245
Average (Standard dev.)-0.000012395362 (±0.0057468526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 283.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Blood coagulation factor VIII bound to patient-derived anti-C1...

Fileemd_29770_half_map_1.map
AnnotationBlood coagulation factor VIII bound to patient-derived anti-C1 domain antibody inhibitor; half-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Blood coagulation factor VIII bound to patient-derived anti-C1...

Fileemd_29770_half_map_2.map
AnnotationBlood coagulation factor VIII bound to patient-derived anti-C1 domain antibody inhibitor; half-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of coagulation factor VIII bound to a patient-derived a...

EntireName: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
Components
  • Complex: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
    • Protein or peptide: Coagulation factor VIII chimera from human and pig
    • Protein or peptide: NB33 light chain
    • Protein or peptide: NB33 heavy chain

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Supramolecule #1: Structure of coagulation factor VIII bound to a patient-derived a...

SupramoleculeName: Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Coagulation factor VIII (molecule 1) is a chimera of human+pig proteins which was expressed in CHO cells.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Coagulation factor VIII chimera from human and pig

MacromoleculeName: Coagulation factor VIII chimera from human and pig / type: protein_or_peptide / ID: 1
Details: This protein is a chimera of human+pig coagulation factor VIII (PDB: 6MF0).
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 168.287047 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MQLELSTCVF LCLLPLGFSA IRRYYLGAVE LSWDYRQSEL LRELHVDTRF PATAPGALPL GPSVLYKKTV FVEFTDQLFS VARPRPPWM GLLGPTIQAE VYDTVVVTLK NMASHPVSLH AVGVSFWKSS EGAEYEDHTS QREKEDDKVL PGKSQTYVWQ V LKENGPTA ...String:
MQLELSTCVF LCLLPLGFSA IRRYYLGAVE LSWDYRQSEL LRELHVDTRF PATAPGALPL GPSVLYKKTV FVEFTDQLFS VARPRPPWM GLLGPTIQAE VYDTVVVTLK NMASHPVSLH AVGVSFWKSS EGAEYEDHTS QREKEDDKVL PGKSQTYVWQ V LKENGPTA SDPPCLTYSY LSHVDLVKDL NSGLIGALLV CREGSLTRER TQNLHEFVLL FAVFDEGKSW HSARNDSWTR AM DPAPARA QPAMHTVNGY VNRSLPGLIG CHKKSVYWHV IGMGTSPEVH SIFLEGHTFL VRHHRQASLE ISPLTFLTAQ TFL MDLGQF LLFCHISSHH HGGMEAHVRV ESCAEEPQLR RKADEEEDYD DNLYDSDMDV VRLDGDDVSP FIQIRSVAKK HPKT WVHYI AAEEEDWDYA PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV RFMAYTDETF KTREAIQHES GILGPLLYGE VGDTL LIIF KNQASRPYNI YPHGITDVRP LYSRRLPKGV KHLKDFPILP GEIFKYKWTV TVEDGPTKSD PRCLTRYYSS FVNMER DLA SGLIGPLLIC YKESVDQRGN QIMSDKRNVI LFSVFDENRS WYLTENIQRF LPNPAGVQLE DPEFQASNIM HSINGYV FD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRG M TALLKVSSCD KNTGDYYEDS YEDISAYLLS KNNAIEPRSF AQNSRPPSAS APKPPVLRRH QRDISLPTFQ PEEDKMDYD DIFSTETKGE DFDIYGEDEN QDPRSFQKRT RHYFIAAVEQ LWDYGMSESP RALRNRAQNG EVPRFKKVVF REFADGSFTQ PSYRGELNK HLGLLGPYIR AEVEDNIMVT FKNQASRPYS FYSSLISYPD DQEQGAEPRH NFVQPNETRT YFWKVQHHMA P TEDEFDCK AWAYFSDVDL EKDVHSGLIG PLLICRANTL NAAHGRQVTV QEFALFFTIF DETKSWYFTE NVERNCRAPC HL QMEDPTL KENYRFHAIN GYVMDTLPGL VMAQNQRIRW YLLSMGSNEN IHSIHFSGHV FSVRKKEEYK MAVYNLYPGV FET VEMLPS KVGIWRIECL IGEHLQAGMS TTFLVYSKKC QTPLGMASGH IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTK EPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPII ARYI RLHPTHYSIR STLRMELMGC DLNSCSMPLG MESKAISDAQ ITASSYFTNM FATWSPSKAR LHLQGRSNAW RPQVNN PKE WLQVDFQKTM KVTGVTTQGV KSLLTSMYVK EFLISSSQDG HQWTLFFQNG KVKVFQGNQD SFTPVVNSLD PPLLTRY LR IHPQSWVHQI ALRMEVLGCE AQDLY

UniProtKB: Coagulation factor VIII, Coagulation factor VIII, Coagulation factor VIII, Coagulation factor VIII

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Macromolecule #2: NB33 light chain

MacromoleculeName: NB33 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.972436 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDTVT IACRASRDIR NDLAWYQQKP GKAPKLLIYA TSRLQSGVPS RFSGSGSFTD FTLTINSLQP DDSATYYCL QDSDYPLTFG GGTKVDIKGT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS LSASVGDTVT IACRASRDIR NDLAWYQQKP GKAPKLLIYA TSRLQSGVPS RFSGSGSFTD FTLTINSLQP DDSATYYCL QDSDYPLTFG GGTKVDIKGT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNR

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Macromolecule #3: NB33 heavy chain

MacromoleculeName: NB33 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.94566 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG VVQPGRSLRL SCVDSGLTFS SYGMHWVRQA PGAGLEWVAV ISYDGNDKYY ADSVKGRFAI SRDNAKNTLY LQMNSLTIE DTAVYYCAKD LIESNIAEAF WGQGTLVTVS SKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN S GALTSGVH ...String:
EVQLVESGGG VVQPGRSLRL SCVDSGLTFS SYGMHWVRQA PGAGLEWVAV ISYDGNDKYY ADSVKGRFAI SRDNAKNTLY LQMNSLTIE DTAVYYCAKD LIESNIAEAF WGQGTLVTVS SKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN S GALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTATY TCNVDHKPSN TKVDKRV

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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
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC8H18N2O4SHEPES

Details: 150 mM NaCl, 20 mM HEPES pH 7.4
GridModel: EMS Lacey Carbon / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6993 / Average exposure time: 1.565 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8628762
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6mf0

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 65133
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6mf0


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8g6i:
Coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor

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