Hi Eric, If your Phaser results show a high Z-score (> 8) AND high LLG AND your solution packs without clashes AND refines (even though starting R/Rfree is high) AND reproduces density for the model portion AND produces some Fo-Fc density for the missing portion, most probably your solution is correct. Starting R/Rfree can be high, especially given that you have only ~50% of the molecule and which may have structural differences from the target. What you could try out is to manually position the second domain/portion based on the partial density and any other information you may have, draw a mask around this complete molecule and use this mask for further density modification to see if you can improve the density in the missing region. You can try several different molecular masks with different positions of the missing portion. Unfortunately, if that does not help, you will have to resort to experimental phasing. The phasing power from the partial model may not be good enough to get complete phase information for the whole molecule. This should be easier in your case assuming the MR solution is right. Even if you get weak/poor derivatives, you may be able to find heavy atoms sites using the partial MR phases and then combine MR+experimental phases. Regards, Debanu.
________________________________ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eric Liu Sent: Tuesday, July 31, 2007 2:24 PM To: [email protected] Subject: [ccp4bb] how to bring back the missing density for half of the structure Hi All, I would like to get some help from here for a data set I recently worked on. I have been working on a new kinase data set which does not have a close homolog. The data was collected to 2.1A resolution in space group P212121 however the difference between a and b is only 0.5A. If I index the data as P4, Rmerge is increased from 13% to 39%. I used the most close homologs which have about 37% sequence identity as search model for molecular replacement and it seemed I have got the solution by using Phaser with only the c-terminal part of the search model and also a long loop removed. After changed the different residues back to the target protein, the structure was refined to Rfree/R 46% and 43% to 2.1 A resolution. The existing c-terminal structure has well defined density except 25ish residue at the very c-terminal end doesn't have well connected density. Current model contains about 50% of overall target residues. I can see some extented difference density for several residues going to the N-terminal part and also extented density for the C-terminal loop for several residues. I also see tones of not well-conncted difference density in the N-terminal region. There was no sever clashes between molecules after mount all symmetry related molecules. My question is the following: 1. Have I got the correct solution for the molecular replacement? 2. How can I bring back the missing density for the N-terminal residues and the loop region? I would really appreciate any inputs or suggestions. Eric
