SAXS Structural Analysis Workflow

SAXS data reduction, buffer subtraction, and further analysis were performed using BioXTAS RAW version 2.1.1^{107 (link)}. An average of 30 frames prior to an eluted peak was used for buffer subtraction. Protein peaks were also run through evolving factor analysis (EFA) to deconvolute peaks into the individual scattering components where applicable. The forward scattering intensity I(0) and radius of gyration (R_g) were calculated from the Guinier fit. The normalized Kratky plot, pair distance distribution plot, or P(r), and Porod volume (V_P) were calculated using the program GNOM embedded in the BioXTAS RAW software^{86 (link)}. The calculation of theoretical scattering curves for the crystal structure PDB 1GRI was performed using the program CRYSOL, part of the ATSAS software package (version 3.1.0)^{86 (link),87 (link)}. The initial all-atom model of the full-length SH2/SH2 domain-swapped dimer was generated with PyMOL version 2.5.2 using PDB structures 1GRI (full-length GRB2 dimer) and 6ICH (SH2 domain-only dimer)^{60 (link),71 (link),108} . Each chain of the 1GRI dimer was superimposed over one of the SH2 domains comprising the 6ICH dimer. Intermodular linkers were built, N-terminal His-tags added, and missing amino acids inserted using YASARA^{109 (link)}. Reconstruction of the electron density was calculated from SAXS data using the program DENSS version 1.6 embedded in the BioXTAS RAW software^{89 (link)}. The final representative model was colorized and annotated using PyMOL and Chimera version 1.16^{110 (link)}.