Computational protocol: Structural and functional studies of a noncanonical Dicer from Entamoeba histolytica

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Protocol publication

[…] The SeMet-EhRNaseIII194 structure was solved using the Se-Met single-wavelength anomalous diffraction method with the AutoSol program embedded in the PHENIX suit; the Figure of Merit value was 0.49. The program identified six out of the eight incorporated Se atoms and generated an initial model that covered more than 75% of protein residues in the asymmetric unit. The side chains of the residues were manually built based on the electron density map using the graphic program, Coot. The partial model was then refined against the diffraction data using the Refmac5 program embedded in CCP4i. The more complete model of SeMet-EhRNaseIII194 was built based on the improved density map resulting from the refinement. The EhRNaseIII229 and EhRNaseIII229-Mn2+ complex structures were solved by the molecular replacement method using the Phaser program of CCP4i; the SeMet-EhRNaseIII194 structure was used as the search model. The resulting model was refined using Refmac5 and the phenix.refine program of PHENIX. During refinement, 5% of randomly selected data was set aside for free R-factor cross validation calculations. The 2Fo-Fc and Fo-Fc electron density maps were regularly calculated and used as guides for the building of the missing residues. Water molecules were added either automatically or manually using Coot. Sulfate and metal ions were modeled in the refinement until the last few cycles. The Rwork and Rfree were 0.202 and 0.246, 0.207 and 0.240, and 0.197 and 0.240 for the SeMet-EhRNaseIII194, EhRNaseIII229, and EhRNaseIII194-Mn2+ structures, respectively. All the residues were located in the favored or allowed regions of the Ramachandran plot. The detailed structure refinement statistics are summarized in . […]

Pipeline specifications

Software tools PHENIX, Coot, REFMAC5
Application Protein structure analysis
Organisms Entamoeba histolytica, Aquifex aeolicus