The Human RNA Helicase DDX21 Presents a Dimerization Interface Necessary for Helicase Activity

Marcaida M, Kauzlaric A, Duperrex A, Sülzle J, Moncrieffe M, Adebajo D, Manley S, Trono D, Dal Peraro M, iScience 23(12):101811 (2020) DOI

SASDGX9 – Nucleolar RNA helicase 2 (DDX21) fragment 186-620

Nucleolar RNA helicase 2 fragment 186-620
MWexperimental 93 kDa
MWexpected 98 kDa
VPorod 150 nm3
log I(s) 5.55×101 5.55×100 5.55×10-1 5.55×10-2
Nucleolar RNA helicase 2 fragment 186-620 small angle scattering data  s, nm-1
ln I(s)
Nucleolar RNA helicase 2 fragment 186-620 Guinier plot ln 5.55×101 Rg: 4.4 nm 0 (4.4 nm)-2 s2
(sRg)2I(s)/I(0)
Nucleolar RNA helicase 2 fragment 186-620 Kratky plot 1.104 0 √3 sRg
p(r)
Nucleolar RNA helicase 2 fragment 186-620 pair distance distribution function Rg: 4.6 nm 0 Dmax: 16.9 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Nucleolar RNA helicase 2 fragment 186-620 SWISSMODEL model
log I(s)
 s, nm-1
Nucleolar RNA helicase 2 fragment 186-620 DAMMIF model
Synchrotron SAXS data from solutions of Nucleolar RNA helicase 2 (DDX21) fragment 186-620 in 20 mM HEPES, 500 mM NaCl, 10 % Glycerol, 2 mM TCEP, pH 7.5 were collected on the BM29 beam line at the ESRF storage ring (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.099 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 100.00 μl sample at 10 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superose 6 Increase 10/300 column at 20°C. 3000 successive 1 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 DDX21 Core model was built using the PDB 3I32 as template in SwissModel. The N-terminal unstructured tags were added using I-TASSER and the model was submitted to nonlinear Cartesian Normal Mode Analysis (NOLB NMA). Pepsi-SAXS was used to calculate the fit giving a X2 of ~1.3. We then substituted the helicase domain by the crystal structure of this domain (PDB 6l5n) and removed the His-tags.

Nucleolar RNA helicase 2 fragment 186-620 (DDX21-186-620)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   49.0 kDa
 
UniProt   Q9NR30 (186-620)
Sequence   FASTA