On Fri, Mar 25, 2011 at 9:20 PM, Jason
1) Synchrotron Fluorescence scan indicates the heavy atom definitely presents in my protein (my control is a second protein with the same ligand showed no absorbance spectrum)
Keep in mind that a positive fluorescence scan does not necessarily mean that the element of interest is bound and well-ordered in the crystal - it is easy to get an excellent scan without seeing anything in the maps later.
When I load the mtz file to phenix.maps GUI, the mtz label pulldown menu indicates 4 possible choices for the column to use:(1) IMEAN, SIGIMEAN (2) I(+), sigI(+), I(-), sigI(-) merged (3) F(+), sigF(+), F(-), sigF(-) merged (4) F, sigF, Dano SigDano. I have tried the choices of (2) (3) and (4). However, there is barely any anomalous signal at 4sigma, which makes me wondering if something is not right. The first thing coming to my mind is the mtz labels: I(+), sigI(+), I(-), sigI(-) merged. What does merged mean? Could this be the reason? Other issues that could causing the trouble?
The "merged" means that the input data were only partially merged - this usually happens when processing in HKL2000 using the "no merge original index" setting, where the reflections are not merged to the asymmetric unit (anomalous or not). I've never used XDS, but I guess it must do something similar. Phenix doesn't really deal with data like that; it always merges equivalents (while leaving Friedel pairs alone by default). This usually doesn't have any impact on the anomalous signal. If XSCALE has an option to merge the data more completely, this should make the "merged" tag go away. It sounds like your element of interest isn't very well ordered; do you see the rest of the ligand in the normal maps? If you really want to be sure that it's not a data-handling issue, you could try reprocessing in other programs and confirming the result. I don't know if radiation damage could be at fault, but it's always a possibility. -Nat