Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis.

Shah N, Beckham SA, Wilce JA, Wilce MCJ, Nucleic Acids Res 46(6):3169-3186 (2018) PubMed

SASDBJ8 – Probable ATP-dependent RNA helicase DDX58 without CARDs (Delta-CARDs RIG-I) plus 10mer hairpin dsRNA

Probable ATP-dependent RNA helicase DDX58 (without CARDs)
5´ppp 10mer hairpin dsRNA
MWexperimental 88 kDa
MWexpected 87 kDa
VPorod 146 nm3
log I(s) 4.60×10-2 4.60×10-3 4.60×10-4 4.60×10-5
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA small angle scattering data  s, nm-1
ln I(s)
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA Guinier plot ln 4.60×10-2 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA Kratky plot 1.104 0 3 sRg
p(r)
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA pair distance distribution function Rg: 2.8 nm 0 Dmax: 9 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA CORAL model

log I(s)
 s, nm-1
Probable ATP-dependent RNA helicase DDX58 (without CARDs) 5´ppp 10mer hairpin dsRNA DAMMIF model

Synchrotron SAXS data from solutions of Probable ATP-dependent RNA helicase DDX58 without CARDs (Delta-CARDs RIG-I) plus 10mer hairpin dsRNA in 25 mM HEPES, 150 mM NaCl, 2.5 mM MgCl2, 10% glycerol and 1mM DTT, pH 7.4 were collected using size exclusion chromatography (SEC-SAXS) on the SAXS/WAXS camera on the storage ring Australian Synchrotron (Melbourne, Australia) using a Pilatus 1M detector at a sample-detector distance of 1.6 m and at a wavelength of λ = 0.10332 nm (I(s) vs s, where s = 4π sin θ/λ and 2θ is the scattering angle). Data frames (13 x 2 s exposures) were collected through the SEC elution peak at 35.8 min (GE Healthcare Life Sciences Superdex 200 10/300; 0.4 ml/min; 100 μl injection at 8 mg/ml). The data were normalized to the intensity of the transmitted beam and radially averaged; the radially averaged scattering of the solvent-blank was subtracted and the different curves were scaled to yield the final composite scattering curve. The SAXS data were collected at 15 °C.

Probable ATP-dependent RNA helicase DDX58 (without CARDs) (DC-RIG-I)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   79.8 kDa
 
UniProt   O95786 (239-925)
Sequence   FASTA
 
5´ppp 10mer hairpin dsRNA (10bp)
Mol. type   RNA
Olig. state   Monomer
Mon. MW   7.7 kDa
Sequence   FASTA