Computational protocol: Small angle X-ray scattering studies of CTNNBL1 dimerization and CTNNBL1/CDC5L complex

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Protocol publication

[…] SAXS measurements were carried out at the In-vacuum Undulator 20 beamline (4C SAXS II) of the Pohang Accelerator Laboratory (PAL) Korea. The wavelength and beam size were 0.675 Å and 0.2 (V) × 0.6 (H) mm2, respectively. A two-dimensional charge-coupled detector (Mar USA, Inc.) was employed. A sample-to-detector distance of 4.00 m for SAXS was used. The magnitude of the scattering vector, q = (4π/λ) sin θ, was 0.010 Å−1 < q < 0.165 Å−1, where 2θ is the scattering angle and is the wavelength of the X-ray beam source. The scattering angle was calibrated with polyethylene-b-polybutadiene-b-polystyrene (SEBS) block copolymer standard. We used solution sample cells with 10-μm-thick mica windows, a volume of 50 μl, and an X-ray beam path length of 0.8 mm. All scattering measurements were carried out at 4 °C. The SAXS data were collected in five successive frames of 0.1 min each to monitor radiation damage. There were no changes in the scattering patterns with time, i.e., no radiation damage was detected during the scattering measurements. Protein solutions were measured over a small concentration range, 1.0–5.0 mg/ml, in order to obtain good quality scattering data without any interference from protein molecules (i.e., concentration effect). A low concentration range of 1.0–2.0 mg/ml provided high quality scattering data without concentration effect. Each 2D SAXS pattern was circularly averaged from the beam center and normalized to the transmitted X-ray beam intensity, which was monitored with a scintillation counter placed behind the sample. In the case of CTNNBL1, the measurements were taken in solutions with 0, 100, 200, 300, 400, and 500 mM NaCl, after using the solutions as the experiment background. The measurement of mutant were performed in solutions with 100 mM NaCl, after using the solutions as the experiment background. The measured concentrations of the CTNNBL1/CDC5L construct solutions were 1.0–2.0 mg/ml. The scattering of a 40 mM Tris-HCl pH 8.0 buffer solution without NaCl was used as the experimental background to minimize the conformational change effect by NaCl. The Rg,G (radius of gyration) and forward scattering intensity I(0) at zero angle values were estimated from the scattering data using Guinier analysis. The molecular mass (MM) was calculated from the known scattering of water of 0.01670 cm−1 at 4 °C. The pair distance distribution p(r) function was obtained through the indirect Fourier transform method using the program GNOM. [...] To reconstruct the molecular shapes, the ab initio shape determination program DAMMIF was used. For each model reconstruction, five independent models were selected, and the averaged aligned model was filtered at a given cutoff volume using the program DAMAVER. The final models were obtained by imposing P2 symmetry restriction. The SAXS curves were calculated from the atomic models using the program CRYSOL. For comparison of the overall shapes and dimensions, the ribbon diagrams of the atomic crystal models were superimposed onto the reconstructed dummy atom models using the program SUPCOMB. […]

Pipeline specifications

Software tools ATSAS, DAMMIF, CRYSOL
Application Small-angle scattering
Organisms Dipturus trachyderma
Chemicals Sodium Chloride