A General Small-Angle X-ray Scattering-Based Screening Protocol Validated for Protein-RNA Interactions.

Chen PC, Masiewicz P, Rybin V, Svergun D, Hennig J, ACS Comb Sci 20(4):197-202 (2018) Europe PMC

SASDD25 – 1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8

Protein sex-lethal mutant
RNA decaneucleotide UGU8
MWexperimental 24 kDa
MWexpected 47 kDa
VPorod 41 nm3
log I(s) 7.66×101 7.66×100 7.66×10-1 7.66×10-2
Protein sex-lethal mutant RNA decaneucleotide UGU8 small angle scattering data  s, nm-1
ln I(s)
Protein sex-lethal mutant RNA decaneucleotide UGU8 Guinier plot ln 7.67×101 Rg: 2.5 nm 0 (2.5 nm)-2 s2
(sRg)2I(s)/I(0)
Protein sex-lethal mutant RNA decaneucleotide UGU8 Kratky plot 1.104 0 3 sRg
p(r)
Protein sex-lethal mutant RNA decaneucleotide UGU8 pair distance distribution function Rg: 2.6 nm 0 Dmax: 8.9 nm

Data validation


Fits and models


log I(s)
 s, nm-1
1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8 Rg histogram Rg, nm
Protein sex-lethal mutant RNA decaneucleotide UGU8 EOM/RANCH model
Protein sex-lethal mutant RNA decaneucleotide UGU8 EOM/RANCH model
Protein sex-lethal mutant RNA decaneucleotide UGU8 EOM/RANCH model

Synchrotron SAXS data from solutions of a 1:1 mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8 in 50% dilution of protein buffer (10 mM KP, 50 mM NaCl, 10 mM DTT), pH 6 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 = 0.09919 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 1.00 mg/ml was measured at 20°C. 10 successive 0.500 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.

This synchrotron session suffered from misalignment of the beamstop resulting in parasitic scattering at low angles. Note that the uploaded EOM ensemble is chosen as the 2:2-complex with one RNA monomer as fully bound by two Sxl10GS molecules in the canonical position from Protein data bankk (PDB) entry 1B7F.pdb. The second RNA monomer is placed in the canonical position bound to the free RRM2, resulting in three rigid bodies: RRM2+RNA, RRM1+RRM2+RNA, and RRM1.

Protein sex-lethal mutant (Sxl10GS)
Mol. type   Protein
Organism   Drosophila melanogaster
Olig. state   Dimer
Mon. MW   20.4 kDa
 
UniProt   P19339 (123-294)
Sequence   FASTA
 
RNA decaneucleotide UGU8 (UGU8)
Mol. type   RNA
Organism   synthetic construct
Olig. state   Dimer
Mon. MW   3.1 kDa
Sequence   FASTA