Computational protocol: Rbg1–Tma46 dimer structure reveals new functional domains and their role in polysome recruitment

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Protocol publication

[…] Crystals of Rbg1fl–Tma46205–345 complex were obtained by the vapor diffusion method. The drops were setup at 4°C with 1 μl of 60 mg/ml of protein and 1 μl of reservoir solution (2.38 M sodium formate, 0.2–0.5 M sodium citrate pH 6.5). Three dimensional rectangular crystals with typical dimensions 0.3 × 0.05 × 0.02 mm grew in about 2 weeks. The X-ray diffraction data for the native and selenomethionine derivative were collected from single crystals at the beam line ID14-4 () at the European Synchrotron Radiation Facility at Grenoble, France using an ADSC Quantum Q315r CCD detector.The data were indexed and integrated using MOSFLM (iMOSFLM) and scaled with SCALA in the CCP4 suite (). Heavy atom site search and phasing were done using SHARP () and model building/tracing were done using ARP/wARP (). Cycles of manual model building were performed with the program Coot (). Waters were introduced into the model using ARP/wARP program and validated with the electron density maps in Coot. The structures were refined with REFMAC () for isotropic refinement. TLS groups were defined and used for anisotropic refinement. This included 17 groups comprising of Rbg1 (chain A 2–45, 53–125/131–174/233–299, 175–232, 300–369; chain B 2–53, 54–91/98–125/133–174/233–299, 175–232, 300–369) and Tma46 (chain C 214–240, 241–267, 268–282, 302–313, 314–338; chain D 214–240, 241–267, 268–282, 320–336). Superimpositions between the structures were done using the SSM superpose function in Coot and analysis of the electrostatic surface potential was performed using APBS () in Pymol, also used for generating the structure figures (). […]

Pipeline specifications

Software tools iMosflm, CCP4, ARP/wARP, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae, Dipturus trachyderma