EMDB-1583: 3D structure of the C3bB complex provides insights into the activ...

Basic information

• Entry: Database: EMDB / ID: EMD-1583
• Title: 3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase
• Map data: Structure of the C3bB pro-convertase.

Sample

• Sample: C3bB
• Protein or peptide: C3bB

• Keywords: C3bB / complement alternative pathway
• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / negative staining / Resolution: 28.0 Å
• Authors: Torreira E / Tortajada A / Montes T / Rodriguez de Cordoba S / Llorca O
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2009; Title: 3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase.; Authors: Eva Torreira / Agustín Tortajada / Tamara Montes / Santiago Rodríguez de Córdoba / Oscar Llorca / ; Abstract: Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.

History

Deposition	Nov 24, 2008	-
Header (metadata) release	Dec 3, 2008	-
Map release	Apr 1, 2009	-
Update	Oct 31, 2012	-
Current status	Oct 31, 2012	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_1583.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Structure of the C3bB pro-convertase.
• Voxel size: X=Y=Z: 4.2 Å

Density

Contour Level	1: 4.75 / Movie #1: 4.665481
Minimum - Maximum	-3.26826 - 12.711600000000001
Average (Standard dev.)	0.152448 (±0.975135)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -9 -9 -9 Dimensions 72 72 72 Spacing 72 72 72
Cell	A=B=C: 302.4 Å α=β=γ: 90 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	4.2	4.2	4.2
M x/y/z	72	72	72
origin x/y/z	0.000	0.000	0.000
length x/y/z	302.400	302.400	302.400
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-100	-100	-99
NX/NY/NZ	200	200	200
MAP C/R/S	1	2	3
start NC/NR/NS	-9	-9	-9
NC/NR/NS	72	72	72
D min/max/mean	-3.268	12.712	0.152

Supplemental data

Sample components

Entire : C3bB

• Entire: Name: C3bB

Components

• Sample: C3bB
• Protein or peptide: C3bB

Supramolecule #1000: C3bB

• Supramolecule: Name: C3bB / type: sample / ID: 1000 ; Oligomeric state: One monomer of C3b binds to one monomer of Fb; Number unique components: 2
• Molecular weight: Experimental: 270 KDa / Theoretical: 270 KDa

Macromolecule #1: C3bB

• Macromolecule: Name: C3bB / type: protein_or_peptide / ID: 1 / Name.synonym: complement pro-convertase / Number of copies: 1 / Oligomeric state: dimer / Recombinant expression: No
• Source (natural): Organism: Homo sapiens (human) / synonym: Human

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: Details: 25mM TrisHCl pH8.0, 50mM NaCl, 5 mM NiCl2
• Staining: Type: NEGATIVE / Details: 1% uranyl formate
• Grid: Details: 400 mesh Copper/Palladium grid
• Vitrification: Cryogen name: NONE / Instrument: OTHER

Electron microscopy

• Microscope: JEOL 1230
• Electron beam: Acceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.9 mm / Nominal magnification: 50000
• Sample stage: Specimen holder: Eucentric / Specimen holder model: OTHER / Tilt angle max: 35
• Image recording: Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10.5 µm; Details: images scanned with a MINOLTA Dimage Scan Multi Pro scanner at 2400 dpi and averaged to a final 4.2 angstroms pixel at the specimen; Bits/pixel: 16
• Tilt angle min: 0

Image processing

• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 6000

Atomic model buiding 1

Initial model

PDB ID:

2i07

• Refinement: Space: RECIPROCAL