Computational protocol: Evolution of disorder in Mediator complex and its functional relevance

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Protocol publication

[…] Disorder of each amino acid was predicted for all the Mediator subunits using IUPred () and DISOPRED2 (). Protein FASTA sequences were used as input files and the disorder propensity of each amino acid was obtained in a tabular form. Amino acids with a predicted disorder score greater than or equal to 0.5 were considered to be disordered. As consensus results obtained from both the tools, results obtained from the IUPred algorithm detailed in the current study (Supplementary Figures S1–S5). Further, average disorder of each subunit was calculated as a mean of the disorder score of each amino acid constituting the subunit. IDRs were characterized as continuous stretch of at least 30 amino acids with a predicted disorder score above or equal to 0.5 allowing a maximum of three residues long ordered gap (). Gnuplot available at http://www.gnuplot.info was then used to construct bar graphs for qualitative visualization of IDRs in each kingdom. [...] Euclidean distance was calculated between two organisms as a root of the sum of squared difference in average disorder of the corresponding Mediator complex subunits. The distance thus calculated was used to make 146 × 146 distance matrix. where d(X,Y) is the distance between two organisms X and Y, Xi is the average disorder of subunit i in organism X and Yji is the average disorder of the corresponding subunit i in organism Y.The distance matrices were then used as input to construct dendrograms with PHYLIP-3.695 using neighbor-joining method (). The dendrogram was visualized in FigTreeV1.4.0 (available at http://tree.bio.ed.ac.uk/software/figtree/). [...] Four major types of PTMs such as phosphorylation of Ser, Thr and Tyr, N-linked Asn and O-linked proline glycosylation, Lys/Arg methylation and Lys acetylation were analyzed in Mediator subunits of S. cereviseae, A. thaliana, O. saitva subsp. japonica, C. elegans, D. melanogaster, D. rerio, G. gallus and H. sapiens. PTMs were predicted with freely available web tools NetPhos 2.0 (), NetNGlyc 1.0 (), PMeS () and PAIL () with default parameters. A stretch of 30 residues in Mediator complex subunits was considered as PTM hotspot if the fraction of predicted PTM sites in this stretch was between 0.1 and 0.3. [...] The protein–protein recognition and interaction sites were predicted in Mediator subunits of 30 organisms using MoRFpred (). A stretch of at least five amino acids with a score greater than or equal to 0.5 was considered as a potential recognition and binding site. Such stretches were highlighted on multiple sequence alignments constructed using MAFFT () and ALSCRIPT (). MoRF was called ‘conserved’ if it is aligned in the multiple sequence alignment for at least four organisms. [...] Homology models of AtMed7, AtMed21 and AtMed31 were constructed by submitting FASTA sequences to Phyre2 available at www.sbg.bio.ic.ac.uk/phyre2 (). Quality of model was assessed using PROCHECK (). Modeller was also used to build Arabidopsis Mediator subunit models (). PyMOL was used to align the corresponding models thus generated (https://www.pymol.org). […]

Pipeline specifications

Software tools IUPred, DISOPRED, Gnuplot, PHYLIP, FigTree
Applications Miscellaneous, Phylogenetics, Protein structure analysis
Diseases Genetic Diseases, Inborn