A unique hetero-hexadecameric architecture displayed by the Escherichia coli O157 PaaA2-ParE2 antitoxin-toxin complex.

Sterckx YG, Jové T, Shkumatov AV, Garcia-Pino A, Geerts L, De Kerpel M, Lah J, De Greve H, Van Melderen L, Loris R, J Mol Biol 428(8):1589-603 (2016) Europe PMC

SASDAB9 – EcPaaA2-HisEcParE2 construct

Plasmid stabilization protein ParE
Uncharacterized protein (Antitoxin)
MWI(0) 100 kDa
MWexpected 169 kDa
VPorod 166 nm3
log I(s) 1.75×10-1 1.75×10-2 1.75×10-3 1.75×10-4
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) small angle scattering data  s, nm-1
ln I(s)
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) Guinier plot ln 1.75×10-1 Rg: 3.3 nm 0 (3.3 nm)-2 s2
(sRg)2I(s)/I(0)
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) Kratky plot 1.104 0 √3 sRg
p(r)
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) pair distance distribution function Rg: 3.4 nm 0 Dmax: 15.3 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) CRYSOL model
log I(s)
 s, nm-1
Plasmid stabilization protein ParE Uncharacterized protein (Antitoxin) DAMMIN model
Synchrotron SAXS data from solutions of EcPaaA2-HisEcParE2 construct in 50 mM Tris-HCl 500 mM NaCl, pH 7.5 were collected on the SWING beam line at the SOLEIL storage ring (Saint-Aubin, France) using a AVIEX PCCD170170 detector at a sample-detector distance of 1.8 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 1 and 10 mg/ml were measured at 10°C. 750 successive 0.750 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

Use of SAXS to validate the oligomer state of the obtained crystal structure and to model missing afinity tags

Plasmid stabilization protein ParE (EcParE2)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Octamer
Mon. MW   12.7 kDa
 
UniProt   A0A0D7C2L1 (2-92)
Sequence   FASTA
 
Uncharacterized protein (Antitoxin) (EcPaaA2)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Octamer
Mon. MW   8.5 kDa
 
UniProt   A0A0F6F6Q9 (14-75)
Sequence   FASTA