Computational protocol: Small but versatile: the extraordinary functional and structural diversity of the β-grasp fold

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Protocol publication

[…] The non-redundant (NR) database of protein sequences (National Center for Biotechnology Information, NIH, Bethesda, MD) was searched with the BLASTP program []. Profile searches were conducted using the PSI-BLAST program [] with either single sequences or multiple alignments as queries, with a profile inclusion expectation (e) value threshold of 0.01; searched were iterated until convergence. Hidden Markov models (HMMs) built from alignments using the hmmbuild program were also employed in searches carried out using the hmmsearch program from the HMMER package []. For queries and searches containing compositionally biased segments, the statistical correction option built into the BLAST program was used []. Multiple alignments were constructed using the MUSCLE [] and/or T-COFFEE programs [], followed by manual adjustment based on PSI-BLAST hsp results and information provided by solved three-dimensional structures. All large-scale sequence and structure analysis procedures were carried out with the TASS software package (V. Anantharaman, SB and LA, unpublished results), a successor to the SEALS package []. Protein structures were visualized using the Swiss-PDB viewer [] and cartoons were constructed with the PyMOL program []. Protein secondary structure predictions were made with the JPRED program [], using multiple alignments as queries. Phylogenetic analysis was carried out using a variety of methods including maximum-likelihood, neighbor-joining, and minimum evolution (least squares) methods. Maximum-likelihood distance matrices were constructed using the TreePuzzle 5 program [] and were used as input for the construction of neighbor-joining with the Weighbor program []. Additionally, trees were constructed using the neighbor-joining and minimum evolution methods as implemented in the MEGA program [] and the Bayesian inference method using Markov chain Monte Carlo simulations implemented by the MRBAYES program [].Structure similarity searches were conducted using the standalone version of the DALI program [,] with the query structures scanned against local current version PDB that has all chains as separate entries. The structural hits for each query was collected, even if the DALI Z-score for the match was less than 2.0 and parsed for topological congruence to the β-GF template (Table ) using a custom PERL script. To assess topologically congruence, coordinates of the matching regions detected by DALI searches using known β-GF domains as queries were extracted and analyzed for secondary structure using DSSP program. These secondary structure elements were then represented as a string (corresponding to a row in table ) along with the polarity of the secondary structure element determined from the DALI match to the query structure. These strings were then matched with the equivalent secondary structure pattern strings constructed of bona fide β-grasp domains. If a complete match was obtained these structures were tagged as congruent, while those which were not were ranked in descending order of elements that did not match. This discrimination of the potential candidates was further confirmed by visual examination of each structure. The interacting residues of various proteins of β-grasp fold with their interacting molecules have been deduced using custom made PERL scripts. The scripts encode interacting distance cut-off values of 5.0 Å and 3.5 Å between appropriate atoms in 3-D for deducing the hydrophobic and polar interactions respectively. These inferred interactions were further examined manually using Swiss-PDB viewer for confirming the contacts between amino-acid residues of β-grasp fold proteins and atomic groups of interacting partners. […]

Pipeline specifications

Software tools BLASTP, HMMER, MUSCLE, T-Coffee, Swiss-PdbViewer, PyMOL, JPred, MEGA, MrBayes
Applications Phylogenetics, Nucleotide sequence alignment
Diseases Bacterial Infections
Chemicals Sulfur, Glutamic Acid