Hi
This is all good advice, but there is more that you could do if
you're desperate to use these images.
Having made sure that your spots on the images are not actually
overlapping (i.e. by looking closely at the images), but just flagged
as overlapping in iMosflm, you may be able (i.e. no guarantees) to
persuade Mosflm to use them by -
(i) go to "Processing Options->Advanced Integration" and increase the
values for the Profiles->Tolerance. There's a tool-tip that becomes
visible if you hover the mouse cursor over the entry boxes which
gives more advice on values.
(ii) if you're *really* desperate, and you have noticed that the
overall box width and box height has increased to values much bigger
than your spots (e.g. your spots are really only ~5-10 pixels across,
but the box size has increased to ~30) you could try setting the
overall box size to ~the separation distance in pixels and UNchecking
the "Optimise overall box size" check box - this will fix the overall
dimensions but allow the spot size within it to optimise. This might
get you out of a hole...
As Zhije says, the proper solution is to collect the data without
overlaps, though. Either of the above steps will reduce the
measurement quality, though (i) is better than (ii).
A rule of thumb is to set the crystal to detector distance (in mm) to
at least [maximum cell edge(Å)/wavelength(Å)] (pedants might want to
multiply the RHS of that by 1mm. There are better methods than rules
of thumb, though, e.g. the strategy options that are now widely
available.
On 14 May 2012, at 04:48, Zhijie Li wrote:
Hi Xinghua,
The total intensity of each reflection needs to be accurately
quantitated in order to calculate the structure factors. Not only
the dots need to be well separated in the 3D reciprocal space, but
also a small area around the dots are often needed to calculate the
background for subtraction. That is why when two dots are getting
too close, the programs will reject both dots. The first thing you
need to do is to inspect the images reported with large number of
overlaps to see if the dots are really overlapping or just close to
each other. If the dots are barely touching or just too close to
each other, you can manipulate the SEPERATION parameter to force
the program to take the closely spaced spots. But keep in mind that
you may get less accurate integration by doing so. If many spots
are really touching each other, normally we won't force the
programs to use them. Then the proper remedy is to move the
detector farther and collect the dataset again (also, try to
optimize your freezing to get the mosaicity as low as possible).
For how to play with the mosflm parameters, please read here:
http://www.mrc-lmb.cam.ac.uk/harry/cgi-bin/keyword2.cgi?SEPARATION.
What you need is probably CLOSE.
The hazard of high percentage of overlaps:
If the overlaps are only scattered in a whole dataset, it is OK,
even if they make up 5-10% or even 20% of the whole dataset. It
will only give you a lower completeness, which is not too
detrimental to the structure solution. However, if large,
continuous regions in the dataset are missing, that will cause you
to have poorly defined regions in the calculated map, often seen as
featureless stripes or layers in the map. Unfortunately, when you
have closely spaced reflections, the latter is often the case. The
proper solution is to collect the data at a greater detector
distance to resolve the spots (after taking the test images, both
imosflm and HKL2000 can simulate the collection run to help you to
decide what distance you need). In cases that you have a long unit
cell (>200A), the first thing you need to do is to align the long
edge of the Unix cell with the rotational axis of the pin. In the
difficult cases, you probably even need to shoot multiple crystals
and combine the datasets to get enough completeness.
Zhijie
From: Xinghua Qin
Sent: Sunday, May 13, 2012 10:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] how to ignore spot overlap in imosflm?
Dear CCP4ers,
We collected a diffraction dataset with high percentage of spot
overlaps, It would be so kind to tell me how to ignore spot overlap
in imosflm and explain the hazard of high percentage of spot overlaps.
Thanks in advance.
Best wishes
Xinghua Qin
--
Xinghua Qin
State Key Laboratory of Plant Physiology and biochemistry
College of Biological Sciences
China Agricultural University
No.2, Yuan Ming Yuan West Road
Haidian District, Beijing, China 100193
Tel: +86-10-62732672
E-mail: xinghua...@126.com
Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre,
Hills Road, Cambridge, CB2 0QH
