Computational protocol: Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex*

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Protocol publication

[…] Samples were prepared by passing individual proteins or mixed complexes over a HiLoad 16/60 S200 size exclusion column (GE HealthCare) pre-equilibrated with a buffer that contained 25 mm Tris (pH 7.5), 300 mm NaCl, 1 μm ZnCl2, 5% glycerol, and 3 mm dithiothreitol (DTT). Small angle x-ray scattering data were collected at Cornell High Energy Synchrotron Source G1 beam line (Ithaca, NY) using a 250-μm square x-ray beam with a flux of ∼3 × 1011 photons/s/mm2 at 9.96 keV. Measurements were made at a wavelength of 1.244 Å and at 4 °C using a dual Pilatus 100K-S detector. Data were acquired by taking 10 successive 1–2-s exposures (10–20 s total) with oscillation using 30 μl of sample using two different protein concentrations. Initial processing, including frame averaging and buffer subtraction, was done using the RAW software (). A Guinier approximation was applied to low a q scattering region, and the radius of gyration (Rg) was determined from a linear fit to the Guinier plot (ln(I) versus q2) for the q range that satisfies the relationship qRg <1.3 in the program Primus (ATSAS Package, EMBL) (, ). The pair distance distribution function (P(r)) was calculated using the indirect Fourier transform method in the program GNOM (ATSAS Package, EMBL) (). The maximum protein dimension (Dmax) values were determined from the P(r) analysis, where P(r) approaches zero. We also reported the Rg from the P(r) analysis. Low resolution ab initio envelopes were calculated from the GNOM program outputs with a high resolution limit such that qmax ≤8/Rg using the program DAMMIF (ATSAS Package, EMBL) (). Ten individual models were calculated. The program DAMAVER (ATSAS Package, EMBL) was used to align the 10 models and reject outliers (). Outliers were defined as those models with a normalized spatial discrepancy (NSD) value greater than the average NSD ± 1 standard deviation. At most only a single model was excluded. The aligned models were further refined using the program DAMMIN (ATSAS Package, EMBL) (). Envelope validation was done using the program HydroPro (version 10) to calculate the theoretical sedimentation coefficients of each refined average ab initio model and compared with experimentally determined sedimentation coefficients (). The atomic level shell model with an atomic elements radius value of 4.4 Å was used to calculate sedimentation coefficients (). The buffer density used was 1.0126 g/ml, and the buffer viscosity used was 0.0135 m2/s at 10 °C. The partial specific volume used was calculated based on amino acid sequence. The MLL3 homology model was created using Modeler with the MLL1 crystal structure as a template (PDB code 2W5Z) (, ). The FoxS server was used to predict the solution scattering profile of the MLL3 homology model (). All structural figures were created using the program Chimera (). […]

Pipeline specifications

Software tools ATSAS, DAMMIF, DAMMIN, FoXS
Application Small-angle scattering
Organisms Homo sapiens
Diseases Leukemia