Computational protocol: Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA

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Protocol publication

[…] The full length spUtp24 protein was crystallized at 20 °C by the sitting-drop vapor diffusion method by mixing 1μl of protein sample (10 mg/ml in 10 mM Tris-Cl, pH 8.0 and 400 mM NaCl) with 1μl of reservoir solution (0.2 M calcium acetate and 20% polyethylene glycol 3350). Crystals appeared as cluster after 1 month. Single crystals were separated, cryo-protected in 15% glycerol prepared in the reservoir solution, and flash frozen in liquid nitrogen.Diffraction data were collected at the Shanghai Synchrotron Radiation Facility at beamline BL17U and processed using HKL2000 []. The structure was determined with molecular replacement using the crystal structure of Utp23 as search model in PHENIX []. The model was built in COOT and refined in PHENIX [, ].The crystal belongs to space group C2221 and has one spUtp24 molecule in the asymmetric unit. The final model contains residues 60–192 of spUtp24, one Zn2+ ion, one Ca2+ ion and 14 water molecules. The Ca2+ ion is bound at the crystallographic interface of two molecules and is modeled as half occupancy. The Ca2+ ion is coordinated by Glu128 and His66 of both molecules and not located at the active site of Utp24. […]

Pipeline specifications

Software tools PHENIX, Coot
Application Protein structure analysis
Organisms Schizosaccharomyces pombe, Saccharomyces cerevisiae