An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme

Pierre Dagenais.

SASDKU3 – II-III-VI three-way junction from the Neurospora Varkud Satellite (VS) ribozyme

Neurospora Varkud Satellite ribozyme junction II-III-VI
MWexperimental 22 kDa
MWexpected 20 kDa
VPorod 28 nm3
log I(s) 7.65×100 7.65×10-1 7.65×10-2 7.65×10-3
Neurospora Varkud Satellite ribozyme junction II-III-VI small angle scattering data  s, nm-1
ln I(s)
Neurospora Varkud Satellite ribozyme junction II-III-VI Guinier plot ln 7.65×100 Rg: 2.2 nm 0 (2.2 nm)-2 s2
(sRg)2I(s)/I(0)
Neurospora Varkud Satellite ribozyme junction II-III-VI Kratky plot 1.104 0 3 sRg
p(r)
Neurospora Varkud Satellite ribozyme junction II-III-VI pair distance distribution function Rg: 2.2 nm 0 Dmax: 7.7 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Neurospora Varkud Satellite ribozyme junction II-III-VI PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from solutions of II-III-VI three-way junction from the VS ribozyme in 50 mM MES, 50 mM KCl, 5 mM MgCl2, pH 6.5 were collected on the G1 beam line at the Cornell High Energy Synchrotron Source (CHESS; Ithaca, NY, USA) using a Pilatus 100K detector at a sample-detector distance of 1.5 m and at a wavelength of λ = 0.1245 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 0.5 and 2 mg/ml were measured at 20°C. 10 successive 2 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.

Neurospora Varkud Satellite ribozyme junction II-III-VI (VS ribozyme J236)
Mol. type   RNA
Organism   Neurospora crassa
Olig. state   Monomer
Mon. MW   20.1 kDa
Sequence   FASTA