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- EMDB-2553: Structural analysis of the interaction between C3b complement pro... -

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Basic information

Entry
Database: EMDB / ID: EMD-2553
TitleStructural analysis of the interaction between C3b complement protein and H17(Fab).
Map dataThis is the 3D structure of C3b bound to H17 (Fab)
Sample
  • Sample: C3b
  • Protein or peptide: C3b
  • Protein or peptide: Fab fragment of H17 antibody
KeywordsComplement system / C3b / Fab / H17 antibody
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsPaixao-Cavalcante D / Torreira E / Lindorfer MA / Rodriguez de Cordoba S / Morgan BP / Taylor RP / Llorca O / Harris CL
CitationJournal: J Immunol / Year: 2014
Title: A humanized antibody that regulates the alternative pathway convertase: potential for therapy of renal disease associated with nephritic factors.
Authors: Danielle Paixão-Cavalcante / Eva Torreira / Margaret A Lindorfer / Santiago Rodriguez de Cordoba / B Paul Morgan / Ronald P Taylor / Oscar Llorca / Claire L Harris /
Abstract: Dysregulation of the complement alternative pathway can cause disease in various organs that may be life-threatening. Severe alternative pathway dysregulation can be triggered by autoantibodies to ...Dysregulation of the complement alternative pathway can cause disease in various organs that may be life-threatening. Severe alternative pathway dysregulation can be triggered by autoantibodies to the C3 convertase, termed nephritic factors, which cause pathological stabilization of the convertase enzyme and confer resistance to innate control mechanisms; unregulated complement consumption followed by deposition of C3 fragments in tissues ensues. The mAb, 3E7, and its humanized derivative, H17, have been shown previously to specifically bind activated C3 and prevent binding of both the activating protein, factor B, and the inhibitor, factor H, which are opposite effects that complicate its potential for therapy. Using ligand binding assays, functional assays, and electron microscopy, we show that these Abs bind C3b via a site that overlaps the binding site on C3 for the Ba domain within factor B, thereby blocking an interaction essential for convertase formation. Both Abs also bind the preformed convertase, C3bBb, and provide powerful inhibition of complement activation by preventing cleavage of C3. Critically, the Abs also bound and inhibited C3 cleavage by the nephritic factor-stabilized convertase. We suggest that by preventing enzyme formation and/or cleavage of C3 to its active downstream fragments, H17 may be an effective therapy for conditions caused by severe dysregulation of the C3 convertase and, in particular, those that involve nephritic factors, such as dense deposit disease.
History
DepositionJan 14, 2014-
Header (metadata) releaseFeb 19, 2014-
Map releaseApr 30, 2014-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.45
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7.45
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2553.jpg

Downloads & links

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Map

FileDownload / File: emd_2553.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D structure of C3b bound to H17 (Fab)
Voxel sizeX=Y=Z: 4.56 Å
Density
Contour LevelBy AUTHOR: 7.45 / Movie #1: 7.45
Minimum - Maximum-11.115220069999999 - 37.335979459999997
Average (Standard dev.)0.02826315 (±2.05326891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 328.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.564.564.56
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z328.320328.320328.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-11.11537.3360.028

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Supplemental data

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Sample components

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Entire : C3b

EntireName: C3b
Components
  • Sample: C3b
  • Protein or peptide: C3b
  • Protein or peptide: Fab fragment of H17 antibody

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Supramolecule #1000: C3b

SupramoleculeName: C3b / type: sample / ID: 1000
Details: Sample behaves as a unique peak in gel filtration when analyzed using a Duperdex 200 column.
Oligomeric state: One molecule of C3b bound to one Fab. / Number unique components: 2
Molecular weightExperimental: 230 KDa / Theoretical: 230 KDa / Method: SDS-PAGE

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Macromolecule #1: C3b

MacromoleculeName: C3b / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: plasma
Molecular weightExperimental: 180 KDa / Theoretical: 180 KDa

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Macromolecule #2: Fab fragment of H17 antibody

MacromoleculeName: Fab fragment of H17 antibody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse / Cell: NS0 cells / Location in cell: supernatant
Molecular weightExperimental: 50 KDa
Recombinant expressionRecombinant cell: NS0 cells (murine myeloma cell line)

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 25 mM Tris HCl pH 7.4, 150 mM NaCl, 10 mM DTT
StainingType: NEGATIVE
Details: Grids containing adsorbed protein were floated on 2% uranyl formate for 2 minutes
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1230
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.9 mm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL
DateSep 1, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: EMAN1.9, XMIPP, 2.4 / Number images used: 7227

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Atomic model buiding 1

Initial modelPDB ID:

2i07

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1hod


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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