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Synchrotron SAXS data from solutions of Phospholipase C (PLC H) bound to the chaperone PLC R2 (PLC H/R2 complex) in 25 mM Tris pH 7.5, 100 mM NaCl, 3 mM β-mercaptoethanol were collected on the EMBL P12 beam line at PETRA III (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.12398 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 SAXS data displayed in this entry is a merged scattering profile from three independent SEC-SAXS runs. Three sequential sample injections were performed: 50 µl at 3.5 mgml-1; 50 µl at 7 mgml-1 and 70 µl at 14 mgml-1 onto a Cytiva Superdex 200 Increase 10/300 column at 20°C, at a flow rate of 0.7 mL/min. After normalisation to the intensity of the transmitted beam and subsequent 2D to 1D data reduction (azimuthal averaging) the individual SAXS data frames (1 second exposure; 2100 framed per SEC run) were selected across the respective sample elution peaks and an appropriate region of the elution profile corresponding to SAXS data measured from the solute-free buffer, were identified, averaged and then subtracted to generate individual background-subtracted sample data frames. These data frames underwent further processing, specifically data merging due to the noted effects of X-ray induced radiation damage in the high concentration samples. Briefly, data merging between the high- and low-concentration samples was performed to obtain quality high-angle scattering data from the 14 and 7 mgml-1 datasets that were then combined with the (scaled) low-angle scattering data from the 3.5 mgml-1 sample injection run that qualitatively appeared much-less effected by radiation damage to generate a final merged SAXS profile with contributions from all three SEC-SAXS runs.
The experimental molecular weight was calculated using in-line SEC-MALLS/RI measurements. These MALLS/RI data are included in the full entry zip archive and report the MW results of two independent SEC runs (Run 1 = 106 kDa average MW; Run2 - in line with SAXS 111 kDa). Concentration independent MW estimates calculated directly from the SAXS data place the MW range in the vicinity of 89-99 kDa. All of the SEC-SAXS data and the data used for merging are made available in the full entry zip archive. The DAM models displayed in this entry display a representative individual model and model fit to the SAXS data (top) and the DAMFILT representation (bottom) of the most likely shape obtained from the spatial alignment and subsequent volume and bead occupancy correction of a 10 member DAMMIN model cohort. The results of multiple individual ab initio DAMMIN modelling runs are also provided in the full entry zip archive.
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s, nm-1
