Computational protocol: Functional specialization of domains tandemly duplicated within 16S rRNA methyltransferase RsmC

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Protocol publication

[…] Of the expected seven selenium sites in the asymmetric unit, five were located by the program SOLVE (). The N-terminal, as well as the C-terminal methionine, was disordered. The initial phases were further improved by density modification using Sharp (v 3.0.15) () that improved the overall figure of merit (FOM) to 0.73. The ARP/wARP () built ∼65% of the molecule. The remaining parts of the model were built manually using the program O (). Further cycles of model building alternating with refinement using the program CNS () resulted in the final model, with an R-factor of 0.21 (Rfree = 0.26) to 2.1 Å resolution with no sigma cutoff used during refinement. The final model comprises 334 residues (Ala3-Met336) and 231 water molecules. The N-terminal His-tag and the linker residues were not visible in the electron density map. PROCHECK () analysis shows no residues in the disallowed regions of the Ramachandran plot. A simulated annealing Fo–Fc omit map of the putative SAM-binding site of RsmC is shown (c). Figure 1. [...] Sequence searches were carried out with PSI-BLAST (), and multiple sequence alignment was constructed with MUSCLE (). Sequence conservation was calculated from the sequence alignment and mapped onto the protein structure using ConSurf (). Structure manipulations and modeling was carried out with SwissPDBViewer and PyMol. Structure database searches and superpositions were done with DALI (). […]

Pipeline specifications

Software tools ARP/wARP, CNS, PROCHECK, BLASTP, MUSCLE, ConSurf, PyMOL, DALI
Applications Protein structure analysis, Nucleotide sequence alignment
Organisms Escherichia coli