Dear Deepanshu, Is the primary sequence in the neighbourhood of your missing strands conserved? Perhaps that part differs in your structure relative to the search model. Have you tried omitting the region from MR and rerunning refinement without to see if some new density pops up?
If not, although this doesn’t sound like a classical flexible region, it may still be disordered. If you really can’t build it, you can of course leave it out of the structure and deposit anyway. The rest of the structure might be very relevant for other people. I also note that your refinement R factor is a bit low relative to the Rfree, which is indicating overfitting. Do you use individual B factors? You probably should use grouped Bs at 3 Å. Best, Ditlev --- Ditlev E. Brodersen Lektor, Associate Professor Department of Molecular Biology and Genetics, Aarhus University Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark Phone: +45 21669001 Lab home page: www.bioxray.au.dk/~deb<http://www.bioxray.au.dk/~deb> Educational IT portal: curriculearn.dk<http://curriculearn.dk> Sent from my iPhone Den 10. okt. 2018 kl. 04.59 skrev Deepanshu Choudhary <deepanshui...@gmail.com<mailto:deepanshui...@gmail.com>>: Dear all, I am working on a protein complex. After many efforts, I obtained crystals from one of the refinement screen (which took few weeks to grow) and I got a 3 Å dataset at synchrotron. I scaled the dataset in P21 spacegroup (which is also confirmed by Zanuda and Pointless). There is no twinning detected. I solved the phase using molecular replacement with a model of over 90% sequence identity. After several rounds of refinement with Refmac, the Rfree is 0.307 and Rfactor of 0.23. The density looks good and I can see everything that's important. But one of the proteins has missing density in 2 of its beta strands (corresponding to ~15%) and its not appearing upon several rounds of refinement. Also, the B factors are higher for this protein. The missing beta strands are not at the interface of the complex. I ran some crystals on the gel and did silver staining to find both the proteins and no degradation products. I doubt that there is any proteolytic cleavage because the protein is unlikely to remain folded if those beta strands are chopped out. I want to ask if such a structure with missing density and high B-factors (>100) can be deposited. Is it possible that some parts of the lattice don't have this protein with missing density which is resulting in high B factors? I would appreciate your efforts if someone can send me few references describing such type of structures. I would also welcome any other suggestions and recommendations. Thanks and regards, Deepanshu ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
