Computational protocol: Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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Protocol publication

[…] Sequence searches of the REBASE database of RM systems were done with the BLAST utility at the REBASE website []. Further searches of the GenBank database were carried out at NCBI with PSI-BLAST [] with a conservative e-value threshold of 1E-30. Multiple sequence alignment of TspGWI homologues retrieved from the GenBank was calculated with MUSCLE []. Identification of homology between TspGWI and proteins with known structures was carried out using a new version of the GeneSilico MetaServer [], which is a gateway for a variety of bioinformatic methods, in particular those for the prediction of secondary structure and structurally disordered regions, and also for protein fold-recognition (FR) analysis (an attempt to match the query sequence to known protein structures). Previously, this method has been successfully used to predict structures of several nucleases, for which subsequent crystallographic analyses confirmed the validity of prediction [-]. Alignments between the TspGWI sequence (or its fragments) and sequences of proteins of known structure reported by FR methods were compared, evaluated, and ranked by the PCONS method []. Preliminary structural modelling of individual domains was carried out using the FRankenstein's Monster method [,] and the resulting models were evaluated with MetaMQAP []. […]

Pipeline specifications

Software tools BLASTP, MUSCLE
Databases REBASE
Application Nucleotide sequence alignment
Chemicals Adenosine Triphosphate, S-Adenosylmethionine