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- EMDB-45728: Structure of ecarin from the venom of Kenyan saw-scaled viper in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-45728
TitleStructure of ecarin from the venom of Kenyan saw-scaled viper in complex with the Fab of neutralizing antibody H11
Map dataSharpened consensus map
Sample
  • Complex: Ecarin in complex with neutralizing antibody H11
    • Protein or peptide: Zinc metalloproteinase-disintegrin-like ecarin
    • Protein or peptide: H11 Fab heavy chain
    • Protein or peptide: H11 Fab light chain
    • Protein or peptide: Endogenous peptide
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION
KeywordsSnake venom metalloproteinase / neutralizing antibody / HYDROLASE
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 pyramidum leakeyi (Leakey's carpet viper) / Echis carinatus (saw-scaled viper) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsMindrebo JT / Lander GC
Funding support United States, United Kingdom, 3 items
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, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45728.png

Downloads & links

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Map

FileDownload / File: emd_45728.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened consensus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.57 Å/pix.
x 540 pix.
= 305.64 Å		0.57 Å/pix.
x 540 pix.
= 305.64 Å		0.57 Å/pix.
x 540 pix.
= 305.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.566 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4479696 - 1.9809555
Average (Standard dev.)-0.000012183662 (±0.025619201)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 305.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened consensus map

Fileemd_45728_additional_1.map
AnnotationUnsharpened consensus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_45728_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_45728_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 : Ecarin in complex with neutralizing antibody H11

EntireName: Ecarin in complex with neutralizing antibody H11
Components
  • Complex: Ecarin in complex with neutralizing antibody H11
    • Protein or peptide: Zinc metalloproteinase-disintegrin-like ecarin
    • Protein or peptide: H11 Fab heavy chain
    • Protein or peptide: H11 Fab light chain
    • Protein or peptide: Endogenous peptide
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION

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Supramolecule #1: Ecarin in complex with neutralizing antibody H11

SupramoleculeName: Ecarin in complex with neutralizing antibody H11 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #3-#4, #2
Details: Ecarin was complexed with H11 Fab, which was prepared by papain digestion of an IgG H11 antibody.
Source (natural)Organism: Echis pyramidum leakeyi (Leakey's carpet viper)

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Macromolecule #1: Zinc metalloproteinase-disintegrin-like ecarin

MacromoleculeName: Zinc metalloproteinase-disintegrin-like ecarin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Echis carinatus (saw-scaled viper)
Molecular weightTheoretical: 47.979473 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VPPHERKFEK KFIELVVVVD HSMVTKYNND STAIRTWIYE MLNTVNEIYL PFNIRVALVG LEFWCNGDLI NVTSTADDTL HSFGEWRAS DLLNRKRHDH AQLLTNVTLD HSTLGITFVY GMCKSDRSVE LILDYSNITF NMAYIIAHEM GHSLGMLHDT K FCTCGAKP ...String:
VPPHERKFEK KFIELVVVVD HSMVTKYNND STAIRTWIYE MLNTVNEIYL PFNIRVALVG LEFWCNGDLI NVTSTADDTL HSFGEWRAS DLLNRKRHDH AQLLTNVTLD HSTLGITFVY GMCKSDRSVE LILDYSNITF NMAYIIAHEM GHSLGMLHDT K FCTCGAKP CIMFGKESIP PPKEFSSCSY DQYNKYLLKY NPKCILDPPL RKDIASPAVC GNEIWEEGEE CDCGSPADCR NP CCDAATC KLKPGAECGN GECCDKCKIR KAGTECRPAR DDCDVAEHCT GQSAECPRNE FQRNGQPCLN NSGYCYNGDC PIM LNQCIA LFSPSATVAQ DSCFQRNLQG SYYGYCTKEI GYYGKRFPCA PQDVKCGRLY CLDNSFKKNM RCKNDYSYAD ENKG IVEPG TKCEDGKVCI NRKCVDVNTA Y

UniProtKB: Zinc metalloproteinase-disintegrin-like ecarin

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Macromolecule #2: Endogenous peptide

MacromoleculeName: Endogenous peptide / type: protein_or_peptide / ID: 2
Details: Either degradation product or co-purified with complex. Molecule was not added to the sample.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 613.749 Da
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: H11 Fab heavy chain

MacromoleculeName: H11 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.413074 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSD DTAVYYCARE WDDDDFSFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA PGQGLEWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSD DTAVYYCARE WDDDDFSFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKK

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Macromolecule #4: H11 Fab light chain

MacromoleculeName: H11 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.782082 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YEVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYY CSSYTSSSTL VVFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP V KAGVETTT ...String:
QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YEVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYY CSSYTSSSTL VVFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP V KAGVETTT PSKQSNNKYA ASSYLSLTPE QWKSHKSYSC QVTHEGSTVE KTVAPTECS

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
50.0 mMC4H11NO3Tris
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse but had preferred orientation on holey gold grids. Graphene grid was used to overcome preferred orientation and the two datasets were combined.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Number grids imaged: 2 / Number real images: 5511 / Average exposure time: 5.0 sec. / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 88339 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2147888
CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: OTHER / Details: Ab initio model generated in CryoSPARC
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 334209
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 5
FSC plot (resolution estimation)

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