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Hi Xinghua,

Your completeness is very low and even with a perfect model the quality of your maps will suffer.
Two things may help if you have not already done them:

1) Merge the Friedel pairs. If you have kept F+ and F- apart so far, you could get significantly more complete data by merging them.
2) As has been suggested before, try a three-dimensional profile fitting program (XDS) and give it a very low mosaicity (reflection_range in XDS) of say 0.1-0.2° and do not refine this value. In this way you will only use the central slice of the spots, which is not the optimal method but still much better than discarding the spots altogether. The much more complete data obtained this way should give much better maps.

Best,
Herman

________________________________

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Xinghua Qin
Sent: Tuesday, May 15, 2012 6:39 PM
To: [log in to unmask]
Subject: [ccp4bb] how to ignore spot overlap in imosflm?

Dear all,

Thanks for all of the suggestions, It helps me a lot as I am a newcomer to the structural world.
Increasing the "Profile Tolerance" parameters as Dr Harry Powell has pointed out can increase the completeness by ten percent (from 50% to 60%). I will try other people's advice soon.

Although the completeness is quite low (just 50%), now I have determined the structure (Rfree=0.31, Rfactor=0.24, resolution=2.6). I will look into the map with COOT, and try to get the structure more beautiful.

Thanks again

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: [log in to unmask] <mailto:[log in to unmask]>
<mailto:[log in to unmask]>

At 2012-05-14 11:48:58,"Zhijie Li" <[log in to unmask]> 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: [log in to unmask]
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