Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument.

Bucciarelli S, Midtgaard SR, Nors Pedersen M, Skou S, Arleth L, Vestergaard B, J Appl Crystallogr 51(Pt 6):1623-1632 (2018) Europe PMC

SASDEY4 – Ribonuclease A monomer from in-house SEC-SAXS

Ribonuclease pancreatic
MWexperimental 10 kDa
MWexpected 14 kDa
VPorod 10 nm3
log I(s) 1.66×10-1 1.66×10-2 1.66×10-3 1.66×10-4
Ribonuclease pancreatic small angle scattering data  s, nm-1
ln I(s)
Ribonuclease pancreatic Guinier plot ln 1.66×10-1 Rg: 1.5 nm 0 (1.5 nm)-2 s2
(sRg)2I(s)/I(0)
Ribonuclease pancreatic Kratky plot 1.104 0 3 sRg
p(r)
Ribonuclease pancreatic pair distance distribution function Rg: 1.5 nm 0 Dmax: 4.8 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Ribonuclease pancreatic PDB model

log I(s)
 s, nm-1
Ribonuclease pancreatic DAMFILT model

SAXS data from solutions of Ribonuclease A monomer from in-house SEC-SAXS in PBS, pH 7.4 were collected on a Xenocs BioXolver L instrument at the Copenhagen University, Department of Drug Design and Pharmacology (Copenhagen, Denmark) using a 20Hz Pilatus 300K detector at a sample-detector distance of 0.7 m and at a wavelength of λ = 0.134 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle).

The RNaseA sample (500 microlitres at 7.5 mg/ml) was injected onto a GE Healthcare S200 Increase 10/300 (24 ml) column at a flow rate of 0.1 ml/min at 23°C. A total of 500 x 30 second SAXS data frames were recorded throughout the RNaseA elution. After background solvent corrections, SEC-SAXS data frames 239-249, corresponding to monomeric RNase, were scaled, averaged and binned logarithmically to produce the data displayed in this entry. Additional files, including plots of the selected frames and of I(0), HPLC and exposure cell UV traces and Rg vs. time (determined by AUTORG) are included in the additional files of the full entry zip archive.

Ribonuclease pancreatic (RNase A)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Monomer
Mon. MW   13.7 kDa
 
UniProt   P61823 (27-150)
Sequence   FASTA
 
PDB code   9RAT