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Hi Sampath, You are asking many questions at once. Since I am right now trying to solve a very difficult Se-Met structure, here are some ideas: - Do you have an energy scan on your crystal, showing that there is absorbance at the correct wavelength for Se? If yes, you have proof that there was indeed Se in your crystal; - You describe that you cannot find the Se atom. In theory it is possible that the atom is in the crystal, but not in an ordered fashion and therefore you would not be able to find it. That is theory, I think that in the majority of cases it works fine. You have not told us how the data were collected (synchrotron? wavelength? Inverse beam protocol to optimize SAD?) and whether or not a statistical analysis (with scaleit) tells you if there is a signal there. If you see a signal, then you know Se is present AND ordered. - If you have a MR solution, you can try to use those phases to find the Se atom in a phased anomalous difference map and then (provided that everything is consistent) use a combination of experimental and MR phases; but... - You also did not tell us how you know that your MR solution is correct. Specifically, can you see any features in the structure that are not part of the search model and are sensible? If yes, your solution is useful. If no, you should try omit maps to convince yourself that the MR solution isn't (too) biased and in fact correct - Your statistics given suggest that the MR solution is very poor, with a cons iderable chance that it is not valid. Remember that ~55% R-factor is equivalent to a random solution. If you do refinement and there is no improvement and the statistics are poor, chances are good that the solution wasn't correct in the first place. I worry that you have concluded that since the model fits the density nicely, based on MR, then things must be good, but if you look at Kevin Cowtan's web page with the duck and cat, you will be reminded of the fact that with phases from MR you (almost?) always get nice density from MR, but that does not mean at all that it is correct density. So yes, you should pursue the SAD phases. Remember that the signal will be weak (we cannot judge how weak, depends on the number of Se and size of your problem and the wavelength used), but (good news) you should not have problems with non-isomorphism (since you are not comparing two data sets). The SAD phases, after appropriate density modification, may show you a partial structure - I just tried this for my problem and it did not work for me, but then again, you must try to see if it can be done. Also, assuming that you have native, S-containing protein (as opposed to Se) there is the option of comparing the S- versus Se-protein and you might be able to get phases from that. Finally, others on this forum are better in this matter: 1.6A is fairly high resolution and I wonder if it is possible to pursue direct methods. Probably too low resolution, but I wouldn't know all that w ell, my maps are 5A resolution, so I don't worry about that option. Mark -----Original Message----- From: Sampath Natarajan <[log in to unmask]> To: [log in to unmask] Sent: Fri, 25 Jul 2008 11:05 am Subject: [ccp4bb] Refinement problem Dear all, Now I'm solving a structure with 1.6A resolution. The data seems good with R-sym (12.4) and all other parameters. Actually the data was collected with SAD phasing. When we checked the data we couldn't find the Se atom in the structure. Since the data resolution is good, we tried to do molecular replacement using Balbes program. It was selected a model with 25% sequence identity and we got the good solution too. I could find all residues in the density and also checked the Ramachandran map which shows almost all residues are in the allowed region. The problem is, I have done refinement many times, the R-factor (45.3) and R-free (51.4) is not reducing during the refinement and also figure of merit is not increasing. Still it remains what I got during the first refinement. The density is also not improving much. Also I could find many cuts in the density. My question is…….. 1. Can we use SAD phasing data for MR solution? 2. Is there any other way to reduce the R/R-free? 3. Why the figure of merit is not increasing even after modeled the residues exactly into the electron density? Thanks, Regards, Sampath