[phenixbb] anomalous difference map

Jason phenix.upitt at gmail.com
Sat Mar 26 09:45:29 PDT 2011

On Sat, Mar 26, 2011 at 11:29 AM, Nathaniel Echols <nechols at lbl.gov> wrote:

> On Fri, Mar 25, 2011 at 9:20 PM, Jason <phenix.upitt at gmail.com> wrote:
> > 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.

Indeed, but hopefully it's not the case.

> > 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.

I went through the xds menu again and found that the "merged" seems ok in
terms of anomalous signal.

> It sounds like your element of interest isn't very well ordered; do
> you see the rest of the ligand in the normal maps?

I was hoping to identify the ligand binding position by resolving the
anomalous map first. Shouldn't this be the procedure to locate ligand?

 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 know CCP4 can also generate anomalous difference map. But I myself have
never done it (I googled online and found it not that straight forward). Can
anybody go through it for me please, or there are other handy programs that
can make anomalous difference map?

>  I don't
> know if radiation damage could be at fault, but it's always a
> possibility.
> -Nat

Structural Biology Department
University of Pittsburgh
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