Phase 1:

• The query structure is processed with SPDB, a homemade program, and the amino acid sequence is extracted.
• A second SPDB output is generated and passed to DSSP to extract secondary structure elements, disulfide bridges and solvent accessibility per residue.
  This second SPDB file is also used by CNS to determine non-crystallographic and crystallographic protein:ligand and protein:protein contacts.
• At this point, ENDscript renders a first figure via our ESPript program:
• • Secondary structure elements and residues in alternate confirmation are shown on the top of the sequence query.
  • Accessibility and hydropathy scales, intermolecular contacts and possible disulfide bridges are shown at the bottom.
    
    

Phase 2:

• A BLAST+ search using the sequence of the query structure is performed against a chosen sequence database (PDBAA by default) to detect protein homologues.
• The result is piped to a chosen multiple sequence alignment software (Clustal Omega, MAFFT, MSAProbs or MultAlin).
• A second figure is then generated by ESPript:
• • It shows the aligned sequences colored according to their degree of similarity.
  • In addition, each homologous sequence of known 3D structure is adorned with its secondary structure elements extracted by DSSP.
  • Further information is presented on the bottom alignment as in phase 1.
    
    

Phase 3:

• Two PyMOL session files are generated. They can be downloaded and interactively examined with the molecular 3D visualization program PyMOL installed on the user's computer.

• The first PyMOL file is called "Cartoon":
  This is a ribbon depiction of the query structure colored as a function of the similarity scores calculated from the previous multiple sequence alignment.
  This color ramp from white (low score) to red (identity) allows to quickly locate areas of weak and strong sequence conservation on the query structure.
  A solvent-accessible surface can be mapped with the same coloring code using the PyMOL control panel.
• The second PyMOL file is called "Sausage":
  It shows a variable tube representation of the Cα trace of the query.
  For this drawing, all homologous protein structures have been superimposed on the PDB/CIF query using ProFit, and the size of the tube is proportional to the mean per-residue r.m.s. deviation between Cα pairs.
  The same white-to-red color ramp is used to visualize possible substitutions in sequence.
  This allows the user to identify areas of weak and strong structural conservation and correlate this result with sequence conservation.

• If applicable, these two PyMOL representations can display an assortment of supplementary information compiled by ENDscript:

• Biological assembly (Cα trace style),
• Multiple NMR models (pink Cα trace),
• Disulfide bridges,
• Nucleic acids / ligands / monatomic elements and their contacting residues (ball&stick style),
• Water molecules,
• Strictly conserved residues (magenta ball&stick),
• PDB SITES markers (blue mesh).

• All these features are fully editable by the user thanks to the PyMOL control panel and publication-quality pictures can be rendered quickly.