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Dear BB! I have a strange problem with TLS refinement and refmac. In my AU there are 4 monomers of the same protein. Based on structural subdomains I defined 3 TLS groups for each of those monomers so that the overall TLS group number is 12. When I do TLS and restrained refinement from the GUI using my TLSIN file which has only the group definitions, everything seems fine. I only get quite a few atoms with residul B factor with the strangely exact value of 2.00. Is this normal? I had this also for waters but could overcome this with the keyword "TLSD waters exclude". R factors go down to 18%/22.5% (mild anisiotropic diffraction to 1.8A in the best direction). Now, my real problem is, that when I continue with model building and refinement (restrained with fixed TLS or TLS+restrained), I would like to use the original TLSOUT as the new TLSIN file. When I do so, the R factors rather go up ending with values higher then when leaving out TLS completely (~25%/30%). Can someone explain to me why this happens? I would rather like to avoid rerunning TLS everytime again as it is very time consuming. Any help is greatly appericiated. Matthias PS: I have this issue also for other proteins.