Computational protocol: Linking epigenetic function to electrostatics: The DNMT2 structural model example

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Protocol publication

[…] In silico searches were performed to identify DNMT2 homologous sequences among the 24 sequenced Drosophila genomes () available in the FlyBase database (http://flybase.bio.indiana.edu/blast/). In most cases, the sequence annotated as “DNMT2” was directly recovered. In the other genomes, the D. melanogaster sequence (Flybase Annotation symbol: Dmel/CG10692) was used as query on BLASTn. The D. willistoni sequence used in this study was the isoform B, described by Garcia et al. (2007), which has 341 amino acids. The D. buzzattii and D. suzukii DNMT2 were obtained from the Drosophila buzzatii Genome Project server (http://dbuz.uab.cat/welcome.php) and SpottedWingFlyBase (http://spottedwingflybase.oregonstate.edu/), a dedicated online resource for Drosophila suzukii genomics. All nucleotide and amino acid sequences were then aligned using the Muscle tool []. The Mus musculus DNMT2 sequence (NP_034197) was obtained on BLASTp using as query the human DNMT2 sequence and the Geobacter sulfurreducens DNMT2 directly from accession number (GSU0227) from Uniprot (http://www.uniprot.org/).Using as query sequence of D. melanogaster and human DNMT2, we performed a search through the program BLAST (Basic Local Alignment Search Tool) by homologous structures in the Protein Data Bank (PDB). [...] The DNMT2 models were generated using a homology modelling approach. The Modeller 9.14 [] program was applied using the Spodoptera frugiperda (PDB 4H0N) [] and human DNMT2 structures as templates (PDB 1G55) [], according to the best alignment between the template and the target sequence. We used the complete DNMT2 sequences obtained by searching in silico. The alignment was further verified manually and adjusted, considering the location of insertion/deletion in loops. The homology modelling was performed with a semi-automated approach, using Python scripts previously developed by our group. The modelling protocol followed the default optimization and refinement protocol, as described in the Modeller online manual (available at http://salilab.org/modeller/9.13/manual/node19.html). One hundred models were generated, and the best model was selected using the DOPE score and Procheck [].The generated models were evaluated by Ramachandran plot analysis, which is a well-known evaluation tool to assess the stereochemical quality of a model through the analysis of phi and psi angles for all protein residues and an overall model quality evaluation was performed using Qmean6 [] and Verify3D []. The DNMT2 models were used for subsequent analysis. [...] Sequence and structure analyses are often split into two separate approaches in evolutionary research. In this study, we conducted analyses on the structures of the DNMT2 models generated and their sequences using the Multiseq [] program incorporated in the VMD 1.9.2 [] as a plugin tool. The structural homology measure is based on the structural similarity measure, QH, which was designed to include the effects of the gaps on the aligned portion []. […]

Pipeline specifications

Software tools BLASTN, MUSCLE, BLASTP, MODELLER, PROCHECK, QMEAN, VERIFY3D, VMD
Databases FlyBase
Applications Protein structure analysis, Nucleotide sequence alignment
Organisms Drosophila melanogaster
Chemicals Amino Acids