Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

Bruce HA, Du D, Matak-Vinkovic D, Bandyra KJ, Broadhurst RW, Martin E, Sobott F, Shkumatov AV, Luisi BF, Nucleic Acids Res 46(1):387-402 (2018) PubMed

SASDCZ4 – RNase E 603-850/ATP-dependent RNA helicase (RhlB) binary complex

RNase E 603-850
ATP-dependent RNA helicase RhlB
MWexperimental 139 kDa
MWexpected 77 kDa
VPorod 183 nm3
log I(s) 2.17×10-1 2.17×10-2 2.17×10-3 2.17×10-4
RNase E 603-850 ATP-dependent RNA helicase RhlB small angle scattering data  s, nm-1
ln I(s)
RNase E 603-850 ATP-dependent RNA helicase RhlB Guinier plot ln 2.17×10-1 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
RNase E 603-850 ATP-dependent RNA helicase RhlB Kratky plot 1.104 0 3 sRg
p(r)
RNase E 603-850 ATP-dependent RNA helicase RhlB pair distance distribution function Rg: 5.9 nm 0 Dmax: 29.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
RNase E 603-850 ATP-dependent RNA helicase RhlB GASBOR model

SAXS data (I(s) vs s, where s = 4πsinθ/λ and 2θ is the scattering angle) were collected on the SWING beam ine at the Soleil synchrotron (Gif-sur-Yvette, France). Samples were stored at 288 K in a robotic sample chamber and automatically loaded (22.3 mg/ml) onto a Superdex 200 Increase 3.2/300 gel-filtration column (GE Healthcare) equilibrated with 50 mM Tris HCl pH 7.5, 100 mM NaCl, 100 mM KCl, 10 mM MgCl2, 10 mM DTT and 5 % glycerol (v/v), at a flow rate of 150 μl min-1 by a HPLC (High performance liquid chromatography) instrument (Agilent), directly before elution into the sample detection chamber, where a monochromatic beam illuminated the sample with a wavelength of 0.1022 nm as it flowed through. The sample-detector distance was 1.79 m. During the elution, 250 scattering measurements were taken with 1.5-s time-frames and 0.5-s dead-time between frames. The in-house program FOXTROT (David and Pérez, 2009) was used to normalize and radially average the data. After averaging 20-30 buffer frames in PRIMUS (Konarev et al., 2003), the program DATASW (Shkumatov and Strelkov, 2015) was employed to (i) subtract the buffer average from each sample frame and (ii) calculation of I(0), Rg and MW. Frames corresponding to the peak where Rg was stable were used for averaging.

RNase E 603-850 (RNase E 603-850)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   30.1 kDa
 
UniProt   Q46977 (603-850)
Sequence   FASTA
 
ATP-dependent RNA helicase RhlB (RhlB)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   47.1 kDa
 
UniProt   W8ZZD4 (1-421)
Sequence   FASTA