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Evtrace

How to use

  1. BLAST search

    Search for homologous sequences of the query sequence by BLAST.

    1. Select a data format of query protein in the [Input] field. PDB ID, file in the PDB format, amino acid sequence in the FASTA format and file of amino acid sequence in the FASTA format are accepted.
      PDB ID
      Input PDB ID of the query protein in lowercase. If the PDB ID is found in our database, you can select a chain ID from all chain IDs in the PDB entry.
      PDB format file
      Select the 3D structure file of the query protein in the PDB format.
      FASTA format file
      Select the amino acid sequence file of the query protein in the FASTA format.
      FASTA format text
      Input the amino acid sequence of query protein in the FASTA format.
    2. Modify following BLAST parameters in the [BLAST parameters] field if needed.
      Database
      Database name searched by BLAST
      E-value
      Expectation value threshold
    3. Click [BLAST] to run a BLAST search.
      Command: blastp -query (amino acid sequence of query) -db (database name) -evalue (Expectation value threshold)
  2. Homologous sequences to query data

    Select sequences from the BLAST results and then submit them to the evolutionary trace analysis. In the evolutionary trace analysis, a phylogenetic tree is constructed from the multiple alignment of those sequences by the NJ method, and grouping of sequences is performed on the tree.

    1. BLAST results are shown in the table below (click each ID to view the pairwise alignment of query and subject sequences). Sequences satisfying default conditions for sequence identity (≥20%) and coverage (≥90%) are shown in red and selected with checkboxes initially. Selections can be changed by modifying the conditions on the drop-down lists or check/uncheck each checkbox.
    2. Modify a condition for the evolutionary trace analysis if needed.
      3. Multiple alignment method
      Program for multiple alignment. ClustalW2 or MAFFT can be used.
      Command-line options for clustalW2: -align -infile -outfile
      Command-line options for MAFFT: --auto --clustalout
    3. Click [Exec trace] to perform evolutionary trace analysis.
  3. Results of evtrace

    Results of the evolutionary trace analysis are shown.

    Following parameters for sequence grouping and clustering of trace residues can be changed here.
    Tident
    Cutoff value of identity for sequence grouping. Click a radio button to change the results to those under the corresponding cutoff condition.
    Tdist
    Distance threshold for nearest-neighbor clustering of trace residues. Two clusters are merged if they are closer than this threshold. Click [Change] after entering a value to update results.
    Tresnum
    Number threshold for nearest-neighbor clustering of trace residues. Clusters with the number of residues less than this threshold are ignored. Click [Change] after entering a value to update results.

Description of the result

Following results are shown on each tab.

[Sequence group]
List of sequences with their group ID. All sequences selected in the previous page are grouped using the phylogenetic tree shown in the [Tree] tab. The pairwise alignment of query and subject sequences is shown when the ID is clicked.
[Multiple alignment (group)]
Summary of the multiple alignment obtained by the selected program. Only consensus sequences for each group and the query sequence are shown here. The invariant and class-specific trace resides are shown in pink and gray, respectively. Click [Download raw data] to download the file of the result.
[Multiple alignment (sequence)]
Multiple alignment obtained by the selected program. All sequences and the query sequence are shown here. The invariant and class-specific trace resides are shown in pink and gray, respectively. Click [Download the multiple alignment] to download the file of the result.
[Trace residues (TR)]
Invariant (pink) and class-specific (gray) trace residues on the amino acid sequence and 3D structure.
[Clustering TRs]
Spatial clusters of trace residues on the amino acid sequence and 3D structure. If the trace residue is located at internal position (the surface accessibility < 0.1), the trace residue is not used in the clustering.
[Tree]
Phylogenetic trees of the amino acid sequences.
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