Computational protocol: Crystal Structure of the RNA Recognition Motif of Yeast Translation Initiation Factor eIF3b Reveals Differences to Human eIF3b

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Protocol publication

[…] Needle-shape crystals of eIF3b-RRM76–170 were obtained at 20°C using sitting drop method by mixing equal volumes of protein (11 mg.mL−1) with 30% PEG 4000, 200 mM Li2SO4 and 100 mM Hepes pH 8. Crystals of good diffraction quality grew after three days to the final size of 100*20*10 micrometer. Datasets were collected at the beamline ID23-2 at ESRF, Grenoble. The data set was processed in the space group P222 using XDS and scaled to the final resolution of 1.25 Å. The phase problem was solved by molecular replacement using the crystal structure of Drosophila melanogaster sex-lethal protein (PDB code 3SXL) as the search model in Phaser . The initial model was further built and improved manually in Coot and subsequently subjected to iterative steps of refinement in Phenix and manual model building in Coot.Plate-shape crystals of eIF3b-RRM76–161 were obtained by mixing the same volume of the protein (17 mg.mL−1) with the reservoir containing 33% PEG4000 and 0.1 M Na-citrate (pH 5.6) in sitting drop plates at 20°C. Good diffracting crystals appeared after two weeks. A dataset was collected on the home source beamline equipped with MAR345dtb detector mounted on a Micromax 007 generator operating with a copper target at 1.5417 Å. The dataset was indexed, scaled and reduced using XDS and SCALA in the space group P 222 to the final resolution of 2.6 Å. The structure of eIF3b-RRM76–170 monomer was used as the search model in Phaser. This model was subsequently subjected to iterative steps of refinement in Phenix and manual model building in Coot.Both structures are deposited at the Protein Data Bank with the accession codes 3NS5 (eIF3b-RRM76–161) and 3NS6 (eIF3b-RRM76–170). […]

Pipeline specifications

Software tools XDS, Coot, PHENIX, CCP4
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae, Homo sapiens