Synchrotron SAXS data from solutions of HOTag6 tetramerization domain followed by a 10-Gly linker and monoubiquitin in 20 mM sodium phosphate, 0.5 mM EDTA, 0.02% NaN3, pH 6.8 were collected on the BioCAT 18ID beam line at the Advanced Photon Source (APS), Argonne National Laboratory (Lemont, IL, USA) using a Pilatus3 X 1M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 300.00 μl sample at 4.5 mg/ml was injected at a 0.60 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 2000 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.
HOTag6 is a ~30-amino-acid tetramerization domain followed by a 10-Gly residue linker and then the sequence for monoubiquitin. This is a designed protein (no natural variant exists). The HOTag6 sequence is from the Shu group at UCSF (DOI: 10.1016/j.molcel.2017.12.008).
s, nm-1
