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.

SASDKY3 – Minimal trans Varkud Satellite (VS) ribozyme in 5mM MgCl2

Neurospora Varkud Satellite minimal trans ribozyme
MWexperimental 33 kDa
MWexpected 33 kDa
VPorod 44 nm3
log I(s) 9.18×100 9.18×10-1 9.18×10-2 9.18×10-3
Neurospora Varkud Satellite minimal trans ribozyme small angle scattering data  s, nm-1
ln I(s)
Neurospora Varkud Satellite minimal trans ribozyme Guinier plot ln 9.18×100 Rg: 3.0 nm 0 (3.0 nm)-2 s2
(sRg)2I(s)/I(0)
Neurospora Varkud Satellite minimal trans ribozyme Kratky plot 1.104 0 3 sRg
p(r)
Neurospora Varkud Satellite minimal trans ribozyme pair distance distribution function Rg: 3.1 nm 0 Dmax: 11 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Neurospora Varkud Satellite minimal trans ribozyme PDB (PROTEIN DATA BANK) model

SAXS data from solutions of the minimal trans VS ribozyme in 50 mM MES, 50 mM KCl, 5 mM MgCl2, pH 6.5 were collected using a Xenocs BioXolver L with MetalJet equipped with a Pilatus3 R 300K detector (Université de Montréal, Montréal, Canada) at a wavelength of λ = 0.134 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 0.5 and 0.5 mg/ml were measured at 20°C. 10 successive 30 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.

Neurospora Varkud Satellite minimal trans ribozyme (VS ribozyme TR4P)
Mol. type   RNA
Organism   Neurospora crassa
Olig. state   Monomer
Mon. MW   32.7 kDa
Sequence   FASTA