Saxsess

Holoenzymes were purified as described above and the final buffer composition was 10 mM Mops pH 7, 50 mM NaCl, 2 mM MgCl₂, 1 mM TCEP-HCl and 0.2 mM ATP. Immediately prior to the SAXS experiments purified holoenzyme complexes were concentrated to a final concentration of ~8-12 mg/ml using using 30,000 MWCO Millipore concentrators then filtered using 0.2 micron Z-spin microfuge filters. The SAXS data were collected for 90 min for the wild-type and mutant samples. SAXS data were acquired at 12 °C using the SAXSess (Anton Paar) line collimation (10 mm) instrument at the University of Utah. Data were collected using an image plate detector and normalized buffer subtraction and data reduction to I(q) versus q (q = (4πsinθ)/λ; 2θ is the scattering angle; λ = 1.54 Å CuK_α) was carried out using the program SAXSquant 2.0. P(r) functions for the experimental and theoretical scattering were calculated using GNOM (as implemented in ATSAS 2.5.0; [42 ]). The experimental scattering data were corrected for smearing effects in GNOM using beam length profile parameters measured at the time of protein data collection. The theoretical scattering profile for the Δ366-379 RIα₂:C₂ complex crystal structure was calculated using CRYSOL.