Computational protocol: Steroid Receptor RNA Activator (SRA) Modification by the Human Pseudouridine Synthase 1 (hPus1p): RNA Binding, Activity, and Atomic Model

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Protocol publication

[…] For crystallisation assays, proteins were concentrated to 7 mg/ml, and used for crystallisation after 2 days. We used the sitting drop technique for crystallisation, and mixed the protein with the reservoir solution in a 1∶1 ratio. Crystals of ΔhPus1p appeared after 2–3 days in a buffer containing 30% PEG 4000/glycerol, 0.1 M Bicine/TRIS (pH 8.5), 0.02 M amino acids mixture (0.02 M sodium L-glutamate, 0.02 M DL-alanine, 0.02 M glycine, 0.02 M DL-lysine HCl, 0.02 M DL-serine) at 4°C. The D146A ΔhPus1p mutant crystallised in the following buffer: 20% PEG 8000, 0.1 M TRIS (pH 8.5) at 4°C. Optimized crystallisation conditions are 28% PEG 3350/glycerol, 0.1 M Bicine/TRIS (pH 8.5), and 10% amino acids mixture for the ΔhPus1p and 17% PEG 8000, 0.1 M HEPES (pH 8.0) for the D146A mutant.Crystals were flash cooled in liquid nitrogen after cryo-protection with the reservoir solutions enriched with 10% PEG 400 and 5% glycerol. Data collection from single crystals was performed at ID23-2 at the ESRF (Grenoble, France), and PXIII at the SLS (Villingen, Switzerland). About 20 data sets were collected from single crystals and co-crystals, with only slight variation in the cell parameters. Datasets were integrated and scaled using XDS . Generally, crystals belong to space group P212121 or P22121 with two or one monomer(s) per asymmetric unit, respectively. Datasets suffered from anisotropy and pseudo-translational symmetry, which initially prevented us from solving the structure by molecular replacement. We finally solved the structure with PHASER using Pus1p as a model (pdb id: 4ITS; ).Alternative cycles of maximum likelihood refinement were performed with PHENIX , and model fitting was carried out with COOT . Structures of ΔhPus1p and the ΔhPus1p D146A mutant were refined to 2.0 Å and 2.7 Å resolution, respectively. Crystallographic data are summarized in . Strong positive electron density peaks were observed next to the catalytic residue D146 in ΔhPus1p or the A146 residue in the mutated enzyme. Omit maps, calculated with only the protein models, were used to fit putative ligands found in the crystallisation condition (see ). Anisotropic scaling, bulk solvent correction, and TLS (Translation/Libration/Screw) restraints were used throughout the refinement procedure. Solvent molecules were added last in the unassigned peaks of the Fo-Fc Fourier electron density map. The models show good stereochemistry with no residues in the disallowed region of the Ramachandran plot. The accession number for the coordinates of the structures of ΔhPus1p and D146A ΔhPus1p have been deposited with PDB codes 4NZ6 and 4NZ7, respectively. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Nucleosides, Nucleotides, Pseudouridine, Steroids