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- EMDB-19000: Cryo-EM structure of coagulation factor beta-XIIa in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-19000
TitleCryo-EM structure of coagulation factor beta-XIIa in complex with the garadacimab Fab fragment (asymmetric dimer)
Map dataLocal resolution filtered map
Sample
  • Complex: Ternary complex of coagulation factor beta-XIIa with garadacimab Fab fragment and anti-LC-lambda VHH
KeywordsComplex / coagulation / trypsin-like serine protease / hereditary angioedema (HAE) / BLOOD CLOTTING
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19000.png

Downloads & links

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Map

FileDownload / File: emd_19000.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution filtered map
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.095 Å
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Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.9212264 - 1.1401873
Average (Standard dev.)0.0009757301 (±0.018317135)
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_19000_msk_1.map
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Mask #2

Fileemd_19000_msk_2.map
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Additional map: Unsharpened map

Fileemd_19000_additional_1.map
AnnotationUnsharpened map
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Additional map: Sharpened map

Fileemd_19000_additional_2.map
AnnotationSharpened map
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Half map: Half map B

Fileemd_19000_half_map_1.map
AnnotationHalf map B
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Half map: Half map A

Fileemd_19000_half_map_2.map
AnnotationHalf map A
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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

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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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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
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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: Non-uniform refinement in cryoSPARC was used / Number images used: 17108
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
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC
Details: CryoSPARC 3D Variability Analysis was used to determine the final particle stack for this reconstruction
FSC plot (resolution estimation)

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