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- EMDB-18999: Cryo-EM structure of coagulation factor beta-XIIa in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-18999
TitleCryo-EM structure of coagulation factor beta-XIIa in complex with the garadacimab Fab fragment (symmetric dimer)
Map dataSharpened map
Sample
  • Complex: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
    • Protein or peptide: Coagulation factor XII
    • Protein or peptide: Garadacimab heavy chain variable region
    • Protein or peptide: Garadacimab light chain variable region
  • Ligand: water
KeywordsComplex / coagulation / trypsin-like serine protease / hereditary angioedema (HAE) / BLOOD CLOTTING
Function / homology
Function and homology information


coagulation factor XIIa / plasma kallikrein-kinin cascade / Factor XII activation / Defective SERPING1 causes hereditary angioedema / response to misfolded protein / positive regulation of plasminogen activation / blood coagulation, intrinsic pathway / positive regulation of fibrinolysis / misfolded protein binding / zymogen activation ...coagulation factor XIIa / plasma kallikrein-kinin cascade / Factor XII activation / Defective SERPING1 causes hereditary angioedema / response to misfolded protein / positive regulation of plasminogen activation / blood coagulation, intrinsic pathway / positive regulation of fibrinolysis / misfolded protein binding / zymogen activation / Defective factor XII causes hereditary angioedema / protein autoprocessing / positive regulation of blood coagulation / rough endoplasmic reticulum / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / protein processing / blood coagulation / : / innate immune response / serine-type endopeptidase activity / calcium ion binding / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Coagulation factor XII/hepatocyte growth factor activator / Fibronectin type I domain / Fibronectin, type I / Fibronectin type-I domain signature. / Fibronectin type-I domain profile. / Fibronectin type 1 domain / Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. ...Coagulation factor XII/hepatocyte growth factor activator / Fibronectin type I domain / Fibronectin, type I / Fibronectin type-I domain signature. / Fibronectin type-I domain profile. / Fibronectin type 1 domain / Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. / Fibronectin type-II collagen-binding domain profile. / Fibronectin type 2 domain / EGF-like domain / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / : / Kringle-like fold / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / 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 XII
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsDrulyte I
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2024
Title: Structural basis for the inhibition of βFXIIa by garadacimab.
Authors: Ieva Drulyte / Rajesh Ghai / Saw Yen Ow / Eugene A Kapp / Adam J Quek / Con Panousis / Michael J Wilson / Andrew D Nash / Matthias Pelzing /
Abstract: Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of ...Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.
History
DepositionNov 29, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18999.png

Downloads & links

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Map

FileDownload / File: emd_18999.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 280 pix.
= 306.6 Å		1.1 Å/pix.
x 280 pix.
= 306.6 Å		1.1 Å/pix.
x 280 pix.
= 306.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.095 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-3.281844 - 3.6395345
Average (Standard dev.)0.0007709836 (±0.059126943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 306.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18999_msk_1.map
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Mask #2

Fileemd_18999_msk_2.map
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Additional map: Unsharpened map

Fileemd_18999_additional_1.map
AnnotationUnsharpened map
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Additional map: Local resolution-filtered map

Fileemd_18999_additional_2.map
AnnotationLocal resolution-filtered map
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Half map: Half map A

Fileemd_18999_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_18999_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Ternary complex of coagulation factor beta-XIIa with garadacimab ...

EntireName: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
Components
  • Complex: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
    • Protein or peptide: Coagulation factor XII
    • Protein or peptide: Garadacimab heavy chain variable region
    • Protein or peptide: Garadacimab light chain variable region
  • Ligand: water

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Supramolecule #1: Ternary complex of coagulation factor beta-XIIa with garadacimab ...

SupramoleculeName: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 174 KDa

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Macromolecule #1: Coagulation factor XII

MacromoleculeName: Coagulation factor XII / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.53775 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VVGGLVALRG AHPYIAALYW GHSFCAGSLI APCWVLTAAH CLQDRPAPED LTVVLGQERR NHSCEPCQTL AVRSYRLHEA FSPVSYQHD LALLRLQEDA DGSCALLSPY VQPVSLPSGA ARPSETTLCQ VAGWGHQFEG AEEYASFLQE AQVPFLSLER C SAPDVHGS ...String:
VVGGLVALRG AHPYIAALYW GHSFCAGSLI APCWVLTAAH CLQDRPAPED LTVVLGQERR NHSCEPCQTL AVRSYRLHEA FSPVSYQHD LALLRLQEDA DGSCALLSPY VQPVSLPSGA ARPSETTLCQ VAGWGHQFEG AEEYASFLQE AQVPFLSLER C SAPDVHGS SILPGMLCAG FLEGGTDACQ GDSGGPLVCE DQAAERRLTL QGIISWGSGC GDRNKPGVYT DVAYYLAWIR EH TVSEPEA

UniProtKB: Coagulation factor XII

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Macromolecule #2: Garadacimab heavy chain variable region

MacromoleculeName: Garadacimab heavy chain variable region / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.120281 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLLESGGG LVQPGGSLRL SCAASGFTFS KYIMQWVRQA PGKGLEWVSG IDIPTKGTVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCARA LPRSGYLISP HYYYYALDVW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK D YFPEPVTV ...String:
EVQLLESGGG LVQPGGSLRL SCAASGFTFS KYIMQWVRQA PGKGLEWVSG IDIPTKGTVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCARA LPRSGYLISP HYYYYALDVW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK D YFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPP

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Macromolecule #3: Garadacimab light chain variable region

MacromoleculeName: Garadacimab light chain variable region / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.762174 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVLTQPPSA SGTPGQRVTI SCSGSSSNIG RNYVYWYQQL PGTAPKLLIY SNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC AAWDASLRGV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS ...String:
QSVLTQPPSA SGTPGQRVTI SCSGSSSNIG RNYVYWYQQL PGTAPKLLIY SNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC AAWDASLRGV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS KQSNNKYAAS SYLSLTPEQW KSHRSYSCQV THEGSTVEKT VAPTECS

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 18 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration2 mg/mL
BufferpH: 5.5 / Details: 10 mM Na Acetate, 100 mM NaCl pH 5.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: plasma current 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsImmediately before blotting and plunge freezing, fluorinated octyl maltoside (FOM) was added to the sample to the final concentration of 0.005%-0.01% (w/v)

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Details: Electron source E-CFEG (cold-FEG), energy filter Selectris X
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 2 / Number real images: 4000 / Average electron dose: 50.0 e/Å2 / Details: 2000 with 0.005% FOM and 2000 with 0.01% FOM
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.25 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 271636
Details: Particles were picked from each of the datasets independently using a blob picker using 50-170 A diameter. 124767 particles were picked from the 0.005% FOM dataset and 146869 particles from ...Details: Particles were picked from each of the datasets independently using a blob picker using 50-170 A diameter. 124767 particles were picked from the 0.005% FOM dataset and 146869 particles from the 0.01% FOM dataset.
Startup modelType of model: INSILICO MODEL / In silico model: Ab Initio
Details: Ab Initio reconstruction was performed in cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 135296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non-uniform refinement in cryoSPARC was used
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non-uniform refinement in cryoSPARC was used
FSC plot (resolution estimation)

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