Hi Sampath, Just a couple of other hand-on suggestions about few programs. If you have good completeness upto 1.6A with decent redundancy (>5), experimental phasing is almost always a good idea, depends of course on ordered Se, but hardly ever all of them are disordered, maybe some... how big is the protein? NRes? Matthew Coefficient? Number of Se?
a) Which programs did you use for indexing and scaling? If Denzo/Scalepack, you may consider "no merge original index" and "scale anomalous". Also there is a scenario in Denzo manual which can tell you if you have anomalous signal in case you don't have a scan. Also, pay attention to the space group, hklview and pointless (ccp4 prerelease / Phil Evans) can be quite useful for this, both read mtz files. But NCS with screw axis might make life more difficult. b) Use phenix.xtriage to analyse the data, it indicates if there is a possibility of twining. If it gives twin operators, detwin (in ccp4) can be useful even before you do anything with it. c) Try a few different programs, Solve-Resolve might be the quickest, but SHELXD for finding sites, followed by either AutoSHARP or Crank 1.2 (which uses BP3) and PHASER are good candidates. You may need to compare which works better for a given problem..! I have seen examples where odd di-sulphides create lot of problem in finding Se sites.. and PHASER did very well. d) If the raw phases look good, you may try to give it (F, SigF, FOMM, PHI, FreeR and HLs) directly to Arp/Warp. At 1.6, it might work rather well. For refinement, MLHL or SADL might be very useful. Again, at your kind of resolution, Refmac usually does very well.. HTH, Partha On Fri, Jul 25, 2008 at 6:05 PM, Sampath Natarajan <[EMAIL PROTECTED]> wrote: > 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 > > -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515
