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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: [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 > > 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] > > Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH