Table 1.
Input-output overview for software used in major steps 1–3
| Step(s) | Input | Output of interest | URL (website used in this protocol, for analysis or download) | Particular reason for choosing this program over others for this step (if any) | Notes | |
|---|---|---|---|---|---|---|
| Online application servers, used in analysis or data processing steps | ||||||
| HHpred | 1 | target protein sequence | HHpred fragment recommendation | https://toolkit.tuebingen.mpg.de/tools/hhpred | This step uses HHpred’s ability to identify distantly homologous protein sequences [using HHsearch] | |
| 11a-b | MSA generated in major step 2 (steps 2–10) | HHpred-predicted suitable template ranking (representative groups) + automatically predicted target- template alignment (starting point for further refinement) | https://toolkit.tuebingen.mpg.de/tools/hhpred | For finding template structures for modeling, and to obtain an initial target-template alignment, we prefer to submit a MSA that we carefully checked as starting input | ||
| SWISS-MODEL10 | 14a-b | target-template alignment from step 13g | xyz-coordinate model of modellable target protein fragment following the user- selected template structure and alignment as closely as possible | https://swissmodel.expasy.org | SWISS-MODEL accepts user-provided input and includes bound cofactors into the model that were present in the template if their binding site is conserved | |
| PhyML at NGPhylogeny.fr11,12 | 10a | MSA after steps 2–9 | Phylogenetic tree (for MSA consistency checking) | https://ngphylogeny.fr | PhyML is a widely used maximum-likelihood phylogenetic tree construction method implemented for convenient online use on this platform | |
| iTOL13 | 10a | PhyML tree (Newick format) | Interactive tree visualization | https: //itol.embl.de | ||
| Downloadable applications, used in analysis or data processing steps | ||||||
| ClustalX7 | 3a | target protein and selected homolog sequences | automated MSA (starting point for further refinement) + MSA colored display | http://www.clustal.org/clustal2 | historic and/or personal preference only (original application that introduced ClustalX coloring, with a simple user interface due to fewer options) | ClustalX also offers limited edit functions (but UGENE and Jalview are superior in this aspect). ClustalX is no longer updated therefore not recommended for new users |
| UGENE14 | 4, 5 | automated MSA | MSA after manual edits (removing sequences, trimming, editing) | http://ugene.net | UGENE or Jalview can also be used in step 3a. Both offer many more options than ClustalX i.e., are technically superior examples of alternative routes to generating, visualizing, and editing a protein MSA. | UGENE is a versatile alternative to ClustalX. It offers various alignment algorithms and coloring schemes (inc ClustalX emulation) |
| Jalview8 | 4, 5 | automated MSA | MSA after manual edits (removing sequences, trimming) | https://www.jalview.org | Jalview is a versatile alternative to ClustalX. It offers various alignment algorithms and coloring schemes (inc ClustalX emulation) | |
| UCSF Chimera6 | 12, 14f | multiple template structures + modeled target structure | superimposed bundle of 3D-structures for visual inspection | https://www.rbvi.ucsf.edu/chimera | A successor program is being developed: UCSF ChimeraX | |
| 15c-d | xyz-coordinate model (as returned by SWISS-MODEL) | model for visual inspection after simple practical manipulations (e.g., renumbering of residues, deletion of poorly modeled segments) + the coordinate [.pdb] file that is modified accordingly | https://www.rbvi.ucsf.edu/chimera | |||