SEC–MALS–SAXS data sets were collected using the 18-ID-D BioCAT Beamline at the Advanced Proton Source (APS) at Argonne National Laboratory (Chicago, IL). Samples were centrifuged for 5 min at 13,000 rpm to remove any potential aggregates prior to column loading. Samples containing 4–9 mg/mL of GRB2 WT or mutants in 250 μL were injected onto a 24 mL Superdex 75 Increase 10/300 analytical-grade column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM DTT at a flow rate of 0.5 mL/minute on an Agilent 1300 chromatography system. Column eluant was analyzed in line by the UV absorbance detector of the Agilent 1300 chromatography system, then subsequently directed into the DAWN Heleos-II light scattering (LS) and OptiLab T-rEX refractive index detectors in series. Finally, the elution trajectory directed samples into a 1.0 mm ID quartz capillary SAXS sample cell. Scattering data were collected every 1 s using a 0.5 s exposure and detected with a Pilatus 3 1 M pixel detector (DECTRIS) with a 12 keV (1.033 Å wavelength) X-ray beam covering a q-range of 0.0045 < q < 0.35 Å − 1 (q = 4π/λsinθ, where λ is the wavelength and 2θ is the scattering angle). Accurate protein molecular weights from MALS data were determined using the ASTRA software (Wyatt Technology).
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