Solution and in silico structures using small angle X-Ray scattering and I-tasser

Proteins were exchanged into 20 mM NaH₂PO₄, 50 mM NaCl, pH 7.8 and concentrated between 1–3 mg/mL. X-ray scattering measurements were conducted at the life sciences X-ray scattering (LiX) beamline at the National Synchrotron Light Source II at Brookhaven National Laboratory, Brookhaven, NY. About 10 μl of the enzymes and buffer solutions were continuously flowed through a capillary and exposed to the X-ray beam for 5 s. The measurements were carried out in triplicate. Data processing was performed using an automated Python-based package developed at LiX. Overall, the two-dimensional scattering patterns from protein solutions were first recorded on 3 detectors simultaneously and then converted into one-dimensional scattering profiles. The SAXS/WAXS data were then merged, averaged, and buffer subtracted to obtain relative scattering intensity (I) as a function of the momentum transfer vector, q (q = (4πsinθ)/λ), where λ is the beam wavelength, and θ is the scattering angle. The resulting combined and merged scattering pattern was analyzed using PRIMUS, GNOM, and DAMMIF of the ATSAS software package^21–23. The Rg value derived from the Guinier analysis corresponded well to the Rg obtained through the indirect transform algorithm in GNOM. Theoretical three-dimensional models of the ACPs were generated using the I-TASSER algorithm, which builds protein models from primary sequences by combining remote homologs with ab initio calculations²⁴. Pymol was used for graphical analysis and figure generation.