Dear all, While refining the structure of a protein I’m facing problems with some residues present in the loop region of the protein. There are four residues in four different loops in the protein which is always showing as outliers in Ramachandran plot even after multiple steps of refinement (i.e. even 93% of residues of the refined protein are in preferred region). The way i am refining the loops is by using the module; “fit loop” in Phenix, through which I am fitting residues in single loop followed by refinement, and then reiterating the same process for three more other loops containing the Ramachandran outliers.
The problem I’m facing in the following process is that: after refining a single loop, the corresponding residue within the loop (i.e. Ramachandran outlier) is showing under the preferred region but, after using the refined pdb (i.e. obtained after the first loop refinement ) for second loop refinement, the residue (present in the 1st loop) is again showing as outliers after 2nd refinement, while the residue (i.e. Ramachandran outlier) in 2nd loop is showing within the preferred region. In this way I am only able to fix a single residue (i.e. Ramachandran outlier) in a single loop, or to a maximum of two with a different combinations of refinement (for example: refinement of loop 1 followed by loop 3 instead of loop 2 and so on …) is there any other approach to address the problem ?? Is there any way in which multiple loops can be refined in a single time ?? Any suggestions to address this problem is deeply appreciated. P.S. For identifying the loops in protein i used secondary structure alignment with sequences of similar proteins, whose structures are known and matching with the hitherto refined structure of the protein and the ramachandran outliers are in the middle of the loop. As a novice my sincere apology, if the description of the problem is not clear enough for you to understand.
