Antibody Humanization: Homology Suggestions Dialog Box

Homology Suggestions Dialog Box Features

• Chain options
• Find pattern tools
• Sequence viewer
• Import homologs from option menu
  • Structural Database option and buttons
    • Search Database button
    • Choose Databases button
  • File option and Browse button
  • PDB ID option and Select button
  • Default germline set option and Load button
• Numbering scheme option menu
• Align Homologs button
• Search in options
• Pick Individual Regions button
• Pick Regions by Group button
• Selection by homology criteria section
• Parent structure 3D criteria section

 

Chain options

Select the chain to show in the sequence viewer: the heavy chain or the light chain.

Find pattern tools

Find a particular pattern in a sequence. You can use an extended PROSITE syntax, which makes use of secondary structure and property information; the syntax is explained in the tooltip. See the Multiple Sequence Viewer/Editor — Toolbar topic for more information.

Sequence viewer

The sequences of the selected chain and its homologs are shown in this area, colored by residue type. The secondary structure assignments and disulfide bond linkages are also displayed.

Import homologs from option menu and buttons

Import sequences into the sequence viewer to use for the homology criteria. Only the sequences are needed, no structural information is required. The choices are:

Structural Database option and buttons

Use the current selection of antibody structural databases to find homologs. When you select this option, the following buttons are displayed, to choose and search the database.

Search Database button

Click this button to search the selected antibody databases for homologs. The progress of the search is shown in the status area at the bottom of the panel.

Choose Databases button

Specify the antibody databases to use by clicking Choose Database and locating the databases. You can choose more than one database: this allows you to use your own database as well as the database in the distribution.

File option and Browse button

Read the sequences from a file. When you select this option, the Browse button is displayed. Clicking the button opens a file browser in which you can navigate to the desired location and select the file that contains the sequences. The file can be a FASTA File, a Maestro file, a PDB file, or a plain text file with the sequence.

PDB ID option and Select button

Import sequences from the PDB, by ID. When you select this option, a Select button is displayed. Clicking this button opens the Enter PDB ID dialog box, in which you can enter the PDB IDs of the sequences to import. The sequences are retrieved from a local copy of the PDB if it is available, or from the RCSB web site, depending on the preference set for PDB retrieval.

Default germline set option and Load button

Import sequences from the default human germline set in the software installation. When you select this option, a Load button is displayed. Clicking this button imports the sequences.

Numbering scheme option menu

Choose a numbering scheme for the residues in the antibody. This choice affects the annotation scheme that is used and the definitions of the loops. (Note that the databases are constructed to use the appropriate loop definitions for the numbering scheme chosen.)

Align Homologs button

Perform a multiple sequence alignment of the homologs to the query (parent).

Search in options

Choose the region or regions in which you want to locate residues for mutation, by selecting one or more of the options. By default, the options are for groups of regions, but you can choose individual regions by clicking Pick Individual Regions.

Pick Individual Regions button

Pick Regions by Group button

This button changes between these two alternatives when you click it. Using this button allows you to choose the regions to substitute residues in either by group or by individual region. When you click Pick Individual Regions, options for each region in both chains are displayed in the panel and the options for region groups are dimmed. When you click Pick Regions by Group, the options listed by individual region are hidden again.

Selection by homology criteria options

Set criteria for the selection of residues to mutate based on the homologs found. The variations found between the homologs and the query are used as a basis for choosing default mutations. Each of the criteria that you select is applied, so the residues must meet all specified criteria.

• Variability at position >/< N %—Filter residues based on the percentage variability at the residue position. The percentage variability is the normalized Shannon entropy for residue variation at the position, expressed as a percentage. Choose whether to apply a minimum or a maximum variability from the option menu, and specify the percentage threshold in the text box.

• Variability at position >/< N residue types—Filter residues based on the variation in the residue type at the residue position. The variability is the number of residue types observed at the position. Choose whether to apply a minimum or a maximum variability from the option menu, and specify the threshold for the number of residue types in the text box.

• Ignore positions with group conservations—Ignore (do not select) residues that are strongly conserved or that are either strongly or weakly conserved. Select the appropriate option for strong conservation or both strong and weak conservation. The definitions of strong and weak conservation are those used by ClustalW:
  Strongly conserved groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  Weakly conserved groups: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY.

• Ignore parent sequence in applying above criteria—Ignore the parent (query) sequence when applying the variability or conservation criteria.

• Parent residue different than N % homologs at position—Select residues for which the parent residue is different from more than the specified percentage of homologs at the residue position.

Parent structure 3d criteria options

Set criteria for selection of residues based on the 3D structure of the parent (the structure that was analyzed). The criteria can be based on solvent-accessible surface area (SASA) and interactions between residues. Interactions are determined by a distance cutoff: any residue that has atoms within 4 Å of the side chain is considered to interact.

• Solvent accessible surface area—Select this option to filter residues by their solvent-accessible surface area (SASA) relative to an isolated residue of the same type, and set a threshold for the maximum or minimum allowed relative SASA. This option is useful for locating surface residues. (The SASA is calculated by the Lee-Richards method [12], which is also used in Canvas and Phase).

• Residue side chain makes no more than N interactions with protein—Select this option to filter out residues whose side chains interact with the protein, and set the maximum number of such interactions.

• Residue side chain interacts/does not interact with molecule N—Select this option to filter residues by their interaction with a selected molecule. Choose whether to allow or disallow the interaction from the option menu, and specify the molecule in the text box, or select Pick and pick the molecule in the Workspace.

If you select residues only by their structural attributes, there are no suggestions for the mutations, so the result is that the matching residues are selected in the residues table. You can then set the mutations for these residues. If structural attribute criteria are combined with homology criteria, then the mutations are set as well as selecting the residues.