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

SASDBV8 – Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/ADP-AlFx (SEC-peak2)

5´ppp 8mer hairpin dsRNA
Probable ATP-dependent RNA helicase DDX58 (without CARDs)
MWexperimental 173 kDa
MWexpected 173 kDa
VPorod 230 nm3
log I(s) 2.05×10-2 2.05×10-3 2.05×10-4 2.05×10-5
5´ppp 8mer hairpin dsRNA Probable ATP-dependent RNA helicase DDX58 (without CARDs) small angle scattering data  s, nm-1
ln I(s)
5´ppp 8mer hairpin dsRNA Probable ATP-dependent RNA helicase DDX58 (without CARDs) Guinier plot ln 2.05×10-2 Rg: 3.9 nm 0 (3.9 nm)-2 s2
(sRg)2I(s)/I(0)
5´ppp 8mer hairpin dsRNA Probable ATP-dependent RNA helicase DDX58 (without CARDs) Kratky plot 1.104 0 3 sRg
p(r)
5´ppp 8mer hairpin dsRNA Probable ATP-dependent RNA helicase DDX58 (without CARDs) pair distance distribution function Rg: 4.0 nm 0 Dmax: 16 nm

Data validation


Fits and models


log I(s)
 s, nm-1
5´ppp 8mer hairpin dsRNA Probable ATP-dependent RNA helicase DDX58 (without CARDs) SASREF model

Synchrotron SAXS data from solutions of Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/ADP-AlFx (SEC-peak2) in 25 mM HEPES, 150 mM NaCl, 2.5 mM MgCl2, 10% glycerol and 1mM DTT, 2mM ADP-AlFx, 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 32.7 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.

5´ppp 8mer hairpin dsRNA (8bp)
Mol. type   RNA
Olig. state   Dimer
Mon. MW   6.5 kDa
Sequence   FASTA
 
Probable ATP-dependent RNA helicase DDX58 (without CARDs) (DC-RIG-I)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   79.8 kDa
 
UniProt   O95786 (239-925)
Sequence   FASTA