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- PDB-9clp: Structure of ecarin from the venom of Kenyan saw-scaled viper in ... -

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Basic information

Entry
Database: PDB / ID: 9clp
TitleStructure of ecarin from the venom of Kenyan saw-scaled viper in complex with the Fab of neutralizing antibody H11
Components
  • Endogenous peptide
  • H11 Fab heavy chain
  • H11 Fab light chain
  • Zinc metalloproteinase-disintegrin-like ecarin
KeywordsHYDROLASE / Snake venom metalloproteinase / neutralizing antibody
Function / homology
Function and homology information


peptidase activator activity / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / metalloendopeptidase activity / toxin activity / proteolysis / extracellular region / metal ion binding / plasma membrane
Similarity search - Function
ADAM cysteine-rich / ADAM, cysteine-rich domain / ADAM Cysteine-Rich Domain / Disintegrin, conserved site / Disintegrins signature. / Peptidase M12B, propeptide / Reprolysin family propeptide / Reprolysin domain, adamalysin-type / Reprolysin (M12B) family zinc metalloprotease / Disintegrin ...ADAM cysteine-rich / ADAM, cysteine-rich domain / ADAM Cysteine-Rich Domain / Disintegrin, conserved site / Disintegrins signature. / Peptidase M12B, propeptide / Reprolysin family propeptide / Reprolysin domain, adamalysin-type / Reprolysin (M12B) family zinc metalloprotease / Disintegrin / Disintegrin domain profile. / Homologues of snake disintegrins / Disintegrin domain / Disintegrin domain superfamily / Peptidase M12B, ADAM/reprolysin / ADAM type metalloprotease domain profile. / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Zinc metalloproteinase-disintegrin-like ecarin
Similarity search - Component
Biological speciesEchis carinatus (saw-scaled viper)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsMindrebo, J.T. / Lander, G.C.
Funding support United States, United Kingdom, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM145143 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095892 United States
Wellcome Trust221705/Z/20/Z United Kingdom
CitationJournal: Toxins (Basel) / Year: 2024
Title: Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies.
Authors: Laetitia E Misson Mindrebo / Jeffrey T Mindrebo / Quoc Tran / Mark C Wilkinson / Jessica M Smith / Megan Verma / Nicholas R Casewell / Gabriel C Lander / Joseph G Jardine /
Abstract: Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal ...Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus ) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.
History
DepositionJul 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 11, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2May 14, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Zinc metalloproteinase-disintegrin-like ecarin
C: Endogenous peptide
H: H11 Fab heavy chain
L: H11 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,9748
Polymers94,7884
Non-polymers1864
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Antibody , 2 types, 2 molecules HL

#3: Antibody H11 Fab heavy chain


Mass: 23413.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#4: Antibody H11 Fab light chain


Mass: 22782.082 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)

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Protein / Protein/peptide , 2 types, 2 molecules AC

#1: Protein Zinc metalloproteinase-disintegrin-like ecarin / Snake venom metalloproteinase / SVMP


Mass: 47979.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Echis carinatus (saw-scaled viper) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: Q90495, Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
#2: Protein/peptide Endogenous peptide


Mass: 613.749 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Either degradation product or co-purified with complex. Molecule was not added to the sample.
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ecarin in complex with neutralizing antibody H11 / Type: COMPLEX
Details: Ecarin was complexed with H11 Fab, which was prepared by papain digestion of an IgG H11 antibody.
Entity ID: #1, #3-#4, #2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Echis pyramidum leakeyi (Leakey's carpet viper)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
250 mMTrisC4H11NO31
31
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse but had preferred orientation on holey gold grids. Graphene grid was used to overcome preferred orientation and the two datasets were combined.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 73000 X / Calibrated magnification: 88339 X / Nominal defocus max: 3000 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5511
Image scansWidth: 3840 / Height: 3712 / Movie frames/image: 25

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
8PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2147888
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 334209 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025142
ELECTRON MICROSCOPYf_angle_d0.4676973
ELECTRON MICROSCOPYf_dihedral_angle_d4.113718
ELECTRON MICROSCOPYf_chiral_restr0.04751
ELECTRON MICROSCOPYf_plane_restr0.005914

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