Computational protocol: Functional analysis of archaeal MBF1 by complementation studies in yeast

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Protocol publication

[…] All databases and software used in the present study are publicly available. Sequences of MBF1 and TBP from Archaea and Eukaryotes were retrieved from publicly available databases including Swiss-Prot (sp), GenBank (gb), EMBL (emb), and DDBJ (dbj). A list of Archaea with available complete genome sequence was retrieved from the Genomes On Line Database (GOLD) []. Nucleotide sequences were translated into protein sequences using the TRANSLATE web-server []. Position-Specific Iterated BLAST (PSI-BLAST) searches were performed against the non-redundant (nr) database at the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov[] and were used to identify MBF1, TBP and Gcn4 related sequences. Sequence alignments with E-value less than 10-3 and with bit score greater than 100 were considered significant.The analysis for functional domains was performed using Pfam protein family database http://pfam.sanger.ac.uk/[]. Furthermore, CLUSTAL_X [] with default settings was used to compare MBF1 and TBP sequences. The aligned sequences were inspected and adjusted manually to minimize the number of gaps and insertions. These manual adjustments were based on the sequence similarities, secondary structure prediction, and finally, fold recognition results.The phylogenetic tree was constructed according to the Neighbor-Joining method [] and visualized by MEGA program version 4.0 []. Distance analyses including 1500 bootstrap replicates were performed.Structurally related proteins were identified by searches against known structures in the Protein Data Bank (PDB) [], and for sequence-to-structure alignment of the deduced aMBF1 METASERVER was used []. METASERVER predictions were complemented with those obtained by PHYRE [] and I-TASSER [] for a successful fold recognition approach. The predicted 3 D structure of MBF1 was subjected to a series of tests for evaluating its internal consistency and reliability. Backbone conformation was evaluated by the inspection of the Psi/Phi Ramachandran plot obtained from PROCHECK analysis []. Packing quality of the 3 D model was investigated by the calculation of WHATCHECK Z-score value []. Finally, sequence-structure compatibility was evaluated by VERIFY-3 D []. All these programs were executed from the structure analysis and verification servers' web-site at UCLA http://www.doe-mbi.ucla.edu/. […]

Pipeline specifications

Software tools BLASTP, Clustal W, MEGA, Phyre, I-TASSER, PROCHECK, WHAT_CHECK
Databases DDBJ Pfam UniProt
Applications Phylogenetics, Protein structure analysis
Organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Homo sapiens, Thermoproteus tenax, Methanosarcina mazei, Bombyx mori, Cenarchaeum symbiosum, Nitrosopumilus maritimus