Protein Splicing Panel

Using the Protein Splicing Panel

Protein splicing is performed by taking a segment of one protein and replacing a segment of another protein with the source segment. The proteins are aligned temporarily if they are not already aligned, so that the segment can be simply transferred between the source and the destination. The regions around the attachment points are minimized, but otherwise no change is made to the spliced segment. This is intended to be a quick means of transferring residues from one protein to another.

Prior to splicing a segment of a protein into another protein, you may want to perform a structural alignment of the two proteins, so that the termini of the desired source and destination segments are aligned to each other. This gives you more control over the alignment than the automatic global alignment done as part of the procedure.

Once the source segment is defined, possible attachment points on the (aligned) destination protein are identified. The attachment atoms are the carbonyl carbon on the C-terminus end and the nitrogen on the N-terminus end. Any attachment atoms in the destination that are within 8 Å of the attachment atoms of the source segment are considered as potential attachment points. Of these possible attachment points, the default is selected to preserve the residue numbering if possible, otherwise the attachment atom that forms the shortest peptide bond with the segment is taken. The preservation of the numbering assumes that the splicing is being done between two different structures of the same (or similar) protein, with the same residue numbering.

The splicing is done by temporarily aligning the source protein to the destination if necessary; extracting the source segment and deleting the destination segment (between the attachment points); assigning the destination chain ID to the spliced segment and renumbering the residues to continue from the N-terminal attachment residue numbering. If a duplicate residue number is found, the residue in the spliced segment is assigned a number that is higher than any found in the destination chain. The new segment is attached at the attachment points (or point, for a terminal attachment), and all atoms within 6 bonds of the attachment points are minimized. A new project entry is created for the modified protein.

Apart from the minimization around the attachment points, no attempt is made to relieve strain or clashes with the rest of the target protein. Once the splicing is done, you should run a refinement on the spliced segment and its local environment.

To splice a segment of one protein (the source) into another protein (the destination):

1. Include both proteins in the Workspace.

   You can include more than one protein in the Workspace, but only one protein can be selected as the destination. You might want to do this to identify variable and conserved regions in a set of related proteins, to guide the selection of the segment to splice.

2. If the proteins are already aligned, select Proteins are already structurally aligned to each other.

   This should be done before defining the source segment: once it is defined, the proteins are aligned temporarily if they aren't already.

3. Select a contiguous set of residues in the source protein.

   It is probably easiest to do this in the sequence viewer, which is displayed automatically when you open the panel. If you make the selection in the Workspace, it is filled out to entire resides. You must make sure the residues are attached in a continuous chain.

   Once the residues are selected, the segment selected is reported in the top part of the panel, and the option menus are filled in with the list of possible destination proteins and the residues on the destination to which the splicing could be done.

4. Choose the destination protein, if you have more than two proteins in the Workspace.

5. Change the selection of the attachment residues, if necessary.

   The source segment is attached to these two residues, and the residues between them (but not including them) is deleted.

   If you want to splice the tail of a protein onto another protein, you can choose None as an attachment residue on the C or the N terminal end. You should only do this for one attachment point: setting both to None results in an error message.

6. Click Splice.

   The segment is spliced into the destination protein, and the new protein is added to the project as a new entry and replaces the Workspace. The panel then closes.