Computational protocol: Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

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[…] Data were collected from a single SeMet crystal of MjaL1–mRNA at the MPG/GBF beamline BW6, DESY (Hamburg, Germany) using a MAR CCD detector and were processed and merged with the XDS program suite (). The crystal structure of the regulatory complex MjaL1–mRNA was initially solved by the MAD method in space group I222. An electron density map of a workable quality was obtained and allowed us to build models of both mRNA and L1 molecules within the complex. However, we could not refine this structure to an R-free value less than 40%. Detailed analysis of experimental data shows that crystals of the L1–mRNA complex are pseudohemihedrally twinned and belong to space group C2 with unit cell parameters a = 212.3 Å, b = 68.9 Å, c = 115.9 Å and β = 123.0°, and two monomers in the asymmetric unit. Because for these crystals a × cosβ is almost equal to −c, the cell can be indexed as I222 with a = 68.9, b = 116.1 and c = 178.5 Å, where orthorhombic a-axes are parallel to a* axes of the monoclinic reciprocal unit. The cumulative intensity distribution calculated with TRUNCATE of the CCP4 program suite () indicated pseudohemihedral twinning in space group C2. The twin law h + 2l, −k, −l describes a real space rotation about an axis perpendicular to the crystallographic 2-fold. The twin fraction was estimated to be ∼0.35 by the Britton plot (). The location of the complex in the correct unit cell was done by the molecular replacement method using the obtained structure as a model. The molecular replacement yielded an unambiguous solution with a correlation coefficient of 70.7% and R-factor of 41.2%. Unfortunately, the electron density map calculated with detwinned structure-factor amplitudes and model phases was of lower quality than with twinned data. Therefore, we used twinned data for the model building and refinement. The final model, refined to an R-factor of 27.5% and an R-free of 31.4% at 3.4 Å resolution, includes 214 amino acid residues and 49 nt. Data and refinement statistics are summarized in . Determination of heavy atom positions, initial phasing, density modification and refinement were executed using CNS (). The map interpretation and model building were performed with O (). NCS restraints were used during the early stages of refinement, but the two molecules in the asymmetric unit were finally refined separately. The structural data and the coordinates have been deposited in the Protein Data Bank (accession code 1U63). […]

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