Advanced tools
to further study the Mustguseal alignment





Analysis of multiple alignments of homologous proteins within a superfamily, which are characterized by diverse functional properties within a common structural framework, can reveal the complex interplay between the protein structure, function, and regulation. We recommend sister services of Mustguseal to further study the obtained alignments. Implementation of Mustguseal, Zebra, pocketZebra, and visualCMAT in the laboratory practice can help at studying mechanisms of protein action, designing enzymes with improved properties for practical application and selective modulators of activity of the wild-type proteins.




Understanding how changes in protein sequence affect biological function is one of the most challenging problems of modern structural biology. During evolution of proteins from a common ancestor one functional property can be preserved, while others can vary, leading to functional diversity. For example, homologous enzymes within a superfamily can share a common structural framework and overall reaction chemistry but differ in other catalytic properties such as substrate specificity, enantio- and regioselectivity or even catalyze promiscuous chemical conversions. Why do similar active sites in homologous enzymes perform different chemical transformations? How should we study the structure‒function relationship and predict particular structural changes that lead to functional diversity? On the other side, recent studies have shown that proteins and enzymes, along with quite well-studied functional sites (active sites) and allosteric centers, contain a significant amount of virtually unexplored potential binding pockets. The role of other binding sites, i.e. the majority of existing pockets in this case, remains unknown. How important are these sites for enzyme function? Which binding centers play a physiological role and which can be used to create a protein with new properties for practical applications? How to evaluate the potential role of each specific site for the regulation of protein function?

Analysis of multiple alignments of homologous proteins within a superfamily, which are characterized by diverse functional properties within a common structural framework, can reveal the complex interplay between the protein structure, function, and regulation.

We recommend sister services of Mustguseal to further study the obtained alignments:

  • the Zebra web-service to identify variable amino acid residues responsible for functional diversity and to select hotspots for directed evolution or rational design experiments;
  • the pocketZebra web-service to identify and rank binding sites in proteins by functional significance and select particular positions in the structure that are important for selective binding of substrates/inhibitors/effectors;
  • the visualCMAT web-service to study and interpret correlated mutations/co-evolving residues in protein structures.

Implementation of Mustguseal, Zebra, pocketZebra, and visualCMAT in the laboratory practice can help at studying mechanisms of protein action, designing enzymes with improved properties for practical application and selective modulators of activity of the wild-type proteins.

A submission to Zebra, pocketZebra, and visualCMAT can be made directly from the Mustguseal Results page (see the screen shot below). You can submit the Final Mustguseal alignment to the Zebra web-service (available in Modes 1, 2, and 3), or both the Final Mustguseal alignment and the PDB file of the query protein to Zebra, pocketZebra, and visualCMAT web-services (automatic submission is available only in Mode 1). After pressing the "Submit" button your data will be automatically uploaded from the Mustguseal platform to the selected server and you will be redirected to a corresponding submission page. Press the "Submit" button on that page to start the analysis with the default settings. If you have submitted the task to Mustguseal in Mode 2 or Mode 3 then the automatic submission of the structural data to Zebra, pocketZebra, and visualCMAT web-services will be disabled and you will have to upload the PDB file manually from your computer. Would you wish to customize your submission to Zebra, pocketZebra, and visualCMAT (e.g., use a different PDB file or change the default settings) you can make a submission to the selected server manually (see below).

To make a manual submission to Zebra, pocketZebra, and visualCMAT web-services you will need a multiple protein alignment in fasta format. You can download the Final Mustguseal alignment from the Mustguseal Results page to your computer and then upload it to Zebra, pocketZebra, and visualCMAT web-services. In addition to the Final Mustguseal alignment you may need a PDB structure representing one of the proteins (optionally for Zebra, and mandatory for pocketZebra and visualCMAT). If you have submitted the task to Mustguseal in Mode 1 you would be able to download the representative set of protein PDBs, which were used to build the core structural alignment, by the "Download the core structural alignment" link on the Mustguseal Results page. Choose one PDB from the core collection and upload it to Zebra, pocketZebra, and visualCMAT web-services together with the multiple alignment. If you have submitted the task to Mustguseal in Mode 2 or Mode 3 (i.e., you have constructed the core structural alignment locally on your computer) than you can select any PDB file from the core collection as the representative PDB.




Suplatov, D., Kirilin, E., & Švedas, V. (2016). Bioinformatic Analysis of Protein Families to Select Function-Related Variable Positions. In Understanding Enzymes: Function, Design, Engineering, and Analysis (pp. 351-385) Ed. Allan Svendsen. Pan Stanford.

Suplatov, D., Voevodin, V., & Švedas, V. (2015). Robust enzyme design: Bioinformatic tools for improved protein stability. Biotechnology journal, 10(3), 344-355.

Suplatov, D., & Švedas, V. (2015). Study of functional and allosteric sites in protein superfamilies. Acta Naturae, 7(4), 27, 34-45.