Hi,
I have a problem with the following sentence:
"if I collect all spots I get good map, but it is impossible to solve
the structure by molecular replacement" - if you have a good map (I
assume electron density map) then the structure is solved... for me a
good map is a map I can interpret.
There have been several reports in the literature of structures where
you have "layers" of normal intensities interspersed with layers of very
weak spots in the diffraction patterns. Sometimes with a change in space
group assignment when you go from integrating the strong spots only to
integrating the entire pattern including the very weak intensities. I
don't have a reference off my head right now, some other people on the
bb may have such references available. But differences in spot
intensities (including reassignment of the space group) is described in
Morales et al. (2000), Acta Cryst D56, 1408-1412.
So the reply I can give is the following:
if the diffraction pattern with these strong diffraction intensities
interspersed with weak diffraction intensities only comes from one
crystal, then processing the strong spots only does not explain the
diffraction pattern and is wrong. Similarly with processing the weak
diffraction spots only (which would be difficult to do in practice...
see below). The model produced should explain the diffraction pattern
seen (in terms of space group, layers of molecules arranged in the
crystal...).
If the diffraction pattern originates from 2 crystals (in different
orientations, a case I've had with one large crystal plus a satellite
crystal attached to it in the same loop), it is in principle possible to
integrate only the diffraction spots from only one of the crystals. No
problems for the larger crystal that diffracts more strongly (which is
what I did with my data set - the second lattice was ignored). To
process the diffraction from the smaller crystal would be tricky: you
should have some version of the data frame processing software that
processes first the spots coming from the large crystal and would
produce a copy of the input frames where the optical density
corresponding to the processed reflections is set (on the images, on the
frames) to say 0, for the entire range of frames processed. Then you
would repeat the autoindexing and frame processing (integration) to take
care of the diffraction from the satellite crystal. I don't think such a
modification of the data processing programs is available.
But could you explain more clearly the problem?
Fred.
Marco Lolicato wrote:
Dear all,
I have a particular problem...
so, I have a beautiful crystal with nice diffraction pattern at 2.7A. The
diffraction images are composed by very strong spots and weak spots.
With XDS, if I collect all spots I get good map, but it is impossible to solve
the structure by molecular replacement. If I collect only the strongest spots
(STRONG_PIXEL= 99), I'm able to solve a very good structure...
My problem is: I was trying to get the apo-structure of my protein. I obtained nice crystals of the "apo-protein", but using the method above, in the structure I have found also the ligand!! (probably incorporated during the overexpression).
My protein is a multimer and, biochemically, I found that the endogenus ligand bond to the protein is in the ratio 1:6. ...and I got a crystal in this way.
So, is there a way to analyze all spots in the diffraction pattern to have a
structure of the apo-protein?
Is a good idea discard the strongest spots and try to analyze only the weak
spots? If yes, how I can do it?
All the best,
Marco
