Computational protocol: Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin

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Protocol publication

[…] To identify sequences similar to BXA0138, BXA0139 and BXA0140, we made queries at the National Center for Bioinformatics Information (NCBI) using PSI-BLAST () on the non-redundant database (nr). The nr database was used as a benchmark to select a threshold value for further searches with Jackhmmer from the HMMER3 package ( (). Twilight-zone hits were analyzed with HHpred, Phyre and FFAS (; ; ). As a result, we applied an E-value of 0.01 and performed iterative hmmer (Jackhmmer) searches. The search converged in 4 iterations. All multiple sequence alignments were done using Clustal Omega from the EMBL-EBI Tools ( three-dimensional model of BXA0140 was automatically built with the Phyre program using the structure of a Clostridium difficile bacteriocin, thuricin CD (PDB code 2LA0), as template (). Strong sequence similarity between the template and the analyzed protein is present only in the C-terminal part (see ). For this reason, we applied molecular dynamics to properly fold the protein. We used NAMD () to obtain a stable model of the protein using NPT ensemble (constant pressure and temperature). The simulation was performed for 25 ns in 310 K (minimization and molecular dynamics done alternately) until the stable structure, based on C-alpha carbon RMSD, was reached. We used CHARMM36 force field and set the time step to 1 fs. The protein was placed in the center of a water box with 10 Å layer from the structure and with periodic conditions. In order to stabilize the system NaCl ions were added. Correctness of the model was validated using PROSESS (). The structure was visualized with PyMOL ( Xre binding site was found using the DBTBS database searching tool ( (). When searching a sequence with a PSSM, the score of each subsequence is calculated by adding the contribution of each base in the subsequence according to its position and to the PSSM. To determine if the scored subsequence could be a TFBS (transcription factor binding site), a p-value threshold is used. The p-value here is the probability for a random sequence to have an equal or higher score. When selecting a threshold of 1%, all the subsequences for which this probability is below 0.01 will be returned possible TFBS. […]

Pipeline specifications

Software tools BLASTP, jackhmmer, HHPred, Phyre, FFAS, HMMER, Clustal Omega, NAMD, PROSESS, PyMOL
Applications Protein structure analysis, Amino acid sequence alignment
Organisms Bacillus anthracis