Computational protocol: GyrI-like proteins catalyze cyclopropanoid hydrolysis to confer cellular protection

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[…] The GyrI-like SMBD-containing proteins (totally 12,304 sequences up to September 2016) were obtained using YtkR7 as query from the InterPro website (http://www.ebi.ac.uk/interpro/search/sequence-search). PSI-BLAST using the human TEX264 (NCBI Reference Sequence: NP_001123356.1) as query was performed on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM = blastp&PAGE_TYPE = BlastSearch&LINK_LOC = blasthome) to obtain homologous sequences. All the sequences were first clustered using BLASTClust on the website (https://toolkit.tuebingen.mpg.de/blastclust). The parameters included: (1) sequence length to be covered, 75% and (2) percent identity threshold, 40%. As a result, 907 clusters were obtained, and the representative sequences of the clusters were then used for construction of sequence similarity network (SSN) from the website (http://efi.igb.illinois.edu/efi-est/stepa.php). The network parameters used were: (1) E-value, 5; (2) fraction, 1; and (3) alignment score, 6. [...] Multiple sequence alignment was executed using Clustal Omega on the website (http://www.ebi.ac.uk/Tools/msa/clustalo/), and the alignment results were used to create the sequence logo on the website (http://weblogo.threeplusone.com/create.cgi). [...] The complex structure of lin2189 E157A E185L-YTM was determined by molecular replacement using search model PDB ID 3B49. YTM was added into the structure based on the omit Fo–Fc map and iterative cycles of refinement were carried out using COOT, Refmac, and PHENIX. A random selection of 5% reflections was set aside for cross-validation. PROCHECK and MolProbity were used to access the overall quality of the structural models. All statistics for data collection and structural refinement were listed in Supplementary Table . Structure figures were made using PyMol 1.3. [...] All protein models were prepared in Schrodinger suite software under OPLS_2005 force field. Hydrogen atoms were added to repaired crystal structures according to the physiological pH (7.5) with the PROPKA tool in Protein Preparation tool in Maestro to optimize the hydrogen bond network. Constrained energy minimizations were conducted on the full-atomic models, with heavy atom coverage to 0.4 Å. [...] All ligand structures were produced in Schrodinger Maestro software. The LigPrep module in Schrodinger software was introduced for geometric optimization by using OPLS_2005 force field. The ionization states of ligands were calculated with Epik tool employing Hammett and Taft methods in conjunction with ionization and tautomerization tools. […]

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