Computational protocol: Insights into the molecular architecture and histone H3 H4 deposition mechanism of yeast Chromatin assembly factor 1

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Protocol publication

[…] Size-exclusion chromatography was performed at a flow rate of 0.5 ml min−1 on a Superdex 200 Increase 10/300 GL column equilibrated in SEC-MALLS buffer (20 mM Bis-TRIS, pH 6.5, 500 mM NaCl, 1 mM DTT or TCEP) at 21°C. A 30 µl sample of yCAF1 ± H3-H4 at 2–10 mg ml−1, previously incubated on ice for 10 min to 3 hr, was injected onto the column and multi angle laser light scattering was recorded with a laser emitting at 690 nm using a DAWN-EOS detector (Wyatt TechnologyCorp. Santa Barbara, CA). The refractive index was measured using a RI2000 detector (Schambeck SFD, Germany). The molecular weight was calculated form differential refractive index measurements across the center of the elution peaks using the Debye model for protein using ASTRA software version 6.0.5.3. To verify the stoichiometry of the yCAF1-H3-H4 complex, 1:1 and 1:2 ratios of yCAF1 to H3-H4 dimers were tested.X-ray scattering data were collected using an inline HPLC setup, at the Bio-SAXS beamline (BM29) of the European Synchrotron Radiation Facility (). Inline size-exclusion chromatography was performed at a temperature of 10°C using a Superdex 200 Increase 10/300 GL column equilibrated in SEC-MALLS buffer. Data were collected with a photon-counting Pilatus 1M detector at a sample-detector distance of 2.86 m, a wavelength of λ = 0.991 Å and an exposure time of 1 s/frame. A momentum transfer range of 0.008 to 0.47 Å−1 was covered (q = 4π sinθ/λ, where θ is the scattering angle and λ the X-ray wavelength). Data collected across the peak were subtracted from buffer scattering and the frames showing a constant radius of gyration (Rg) were merged for further analysis. Rg values were obtained from the Guinier approximation sRg <1.3 using Primus (). Distance distribution functions p(r) and the Porod volumes Vp were computed from the entire scattering curve using GNOM (). The program DAMMIN (), was used to generate 40 low-resolution ab initio shape reconstructions. To select the most typical ab initio model of the complex and estimate its possible conformational space, these reconstructions were pairwise aligned and averaged using DAMAVER (). The model with the lowest mean value of normalized spatial discrepancy (NSD) was selected as the most typical reconstruction. To assess the resolution and reliability of the reconstructions we used the Fourier Shell Correlation (FSC) approach as implemented in SASRES (). […]

Pipeline specifications

Software tools ATSAS, DAMMIN
Application Small-angle scattering