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- EMDB-21389: Negative Staining Reconstruction of Tryptase Tetramer bound to E1... -

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Basic information

Entry
Database: EMDB / ID: EMD-21389
TitleNegative Staining Reconstruction of Tryptase Tetramer bound to E104 Fab
Map dataTryptase bound to E104 Fab
Sample
  • Complex: Tryptase bound to E104 Fab
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 15.0 Å
AuthorsKoerber JT / Lazarus B / Maun H / Estevez A / Ciferri C
CitationJournal: Nat Commun / Year: 2020
Title: Bivalent antibody pliers inhibit β-tryptase by an allosteric mechanism dependent on the IgG hinge.
Authors: Henry R Maun / Rajesh Vij / Benjamin T Walters / Ashley Morando / Janet K Jackman / Ping Wu / Alberto Estevez / Xiaocheng Chen / Yvonne Franke / Michael T Lipari / Mark S Dennis / Daniel ...Authors: Henry R Maun / Rajesh Vij / Benjamin T Walters / Ashley Morando / Janet K Jackman / Ping Wu / Alberto Estevez / Xiaocheng Chen / Yvonne Franke / Michael T Lipari / Mark S Dennis / Daniel Kirchhofer / Claudio Ciferri / Kelly M Loyet / Tangsheng Yi / Charles Eigenbrot / Robert A Lazarus / James T Koerber /
Abstract: Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate ...Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.
History
DepositionFeb 15, 2020-
Header (metadata) releaseNov 4, 2020-
Map releaseNov 4, 2020-
UpdateJan 13, 2021-
Current statusJan 13, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0256
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0256
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21389.png

Downloads & links

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Map

FileDownload / File: emd_21389.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTryptase bound to E104 Fab
Voxel sizeX=Y=Z: 1.73 Å
Density
Contour LevelBy AUTHOR: 0.0256 / Movie #1: 0.0256
Minimum - Maximum-0.044255935 - 0.07128593
Average (Standard dev.)0.00021383307 (±0.004442386)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 346.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.731.731.73
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z346.000346.000346.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0440.0710.000

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Supplemental data

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Sample components

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Entire : Tryptase bound to E104 Fab

EntireName: Tryptase bound to E104 Fab
Components
  • Complex: Tryptase bound to E104 Fab

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Supramolecule #1: Tryptase bound to E104 Fab

SupramoleculeName: Tryptase bound to E104 Fab / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 340 kDa/nm

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 6.8 / Details: 10 mM MOPS pH 6.8, 2 M NaCl
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Four microliters of purified beta-tryptase bound to E104.v1 Fab fragment were applied to a freshly glow discharged 400-mesh copper EM grid covered with a thin layer of continuous carbon (Ted ...Details: Four microliters of purified beta-tryptase bound to E104.v1 Fab fragment were applied to a freshly glow discharged 400-mesh copper EM grid covered with a thin layer of continuous carbon (Ted Pella, Redding, CA). After 30 s of incubation, the grids were stained with 5 drops of a freshly prepared 0.75% (w/v) uranyl formate solution.
GridPretreatment - Type: GLOW DISCHARGE / Details: unspecified
Detailspurified through affinity purification and Size Exclusion Chromatography in 10 mM MOPS pH 6.8, 2 M NaCl.

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 40.0 e/Å2

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4a6l

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Number images used: 15000

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