Re: [ccp4bb] Data processing - twinned xtals
Hi Everyone, Just wanted to let everyone know that i was able to process this dataset with XDS ( and lots help from experts !) Thanks again Mahesh On Mon, Aug 19, 2013 at 5:07 PM, Petri Kursula petri.kurs...@oulu.fiwrote: I have often processed images like this with XDS. Of course, you will get a better quality of data with a more optimal strategy, but I would never say never. If I had a penny every time people told me 'you cannot process that'… Petri On Aug 19, 2013, at 11:40 PM, Mahesh Lingaraju mxl1...@psu.edu wrote: Thank you experts for your valuable suggestions. I think Ill try to solve it by proper data collection strategy the next time as i am unable to process my current data even with the tricks that were mentioned here. Thanks again Mahesh On Fri, Aug 16, 2013 at 2:04 PM, Bosch, Juergen jubo...@jhsph.edu wrote: tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine with most SSRL beamlines except of the 12-2 microfocus - but that might have been fixed in the meantime. Jürgen On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote: for #2) I'd suggest get some of those Mitigen loops that are titled. I assume you have hexagonal plates as crystals and you really want to shoot along the thin area of the crystal down the sixfold. With normal loops it's an art to get that crystal to sit upright in the loop but not impossible if you take smaller loops. My longest axis collected was 420 Å to ~2 Å resolution by this method. Jürgen On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote: This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Data processing - twinned xtals
Thank you experts for your valuable suggestions. I think Ill try to solve it by proper data collection strategy the next time as i am unable to process my current data even with the tricks that were mentioned here. Thanks again Mahesh On Fri, Aug 16, 2013 at 2:04 PM, Bosch, Juergen jubo...@jhsph.edu wrote: tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine with most SSRL beamlines except of the 12-2 microfocus - but that might have been fixed in the meantime. Jürgen On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote: for #2) I'd suggest get some of those Mitigen loops that are titled. I assume you have hexagonal plates as crystals and you really want to shoot along the thin area of the crystal down the sixfold. With normal loops it's an art to get that crystal to sit upright in the loop but not impossible if you take smaller loops. My longest axis collected was 420 Å to ~2 Å resolution by this method. Jürgen On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote: This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Data processing - twinned xtals
Hi Mahesh, TESTGEN START 1 END 120 ANGLE 0.5 moving the detector further back or using 2theta and collecting more frames. When you started shooting at your image you should have checked for overlaps ahead of time. I assume this to about 1.8 Å - maybe it would have been wiser to collect a 2.5 Å dataset. Your corners have zero spots when you crank up the contrast, so you are not using all the available detector area, sure the completeness goes down but you might have avoided overlaps like this. BUT not all is lost, if you dare going to XDS you might be able to squeeze sufficient information out of this data set. Good luck, Jürgen On Aug 16, 2013, at 10:38 AM, Mahesh Lingaraju wrote: Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Maheshimage 1.pngimage 2.png .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Data processing - twinned xtals
doesn't necessarily looked twinned to me. Rather it looks like you have a trigonal or hexagonal cell with a long c-axis. On image 2 it seems you may have systematic absences along l, although it is hard to tell the order. Perhaps P31, P32, P62, P64 or spacegroups with these symmetries plus 2-folds perpendicular. On 16 Aug 2013, at 16:38, Mahesh Lingaraju wrote: Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Maheshimage 1.pngimage 2.png
Re: [ccp4bb] Data processing - twinned xtals
PS For future data collections, try to get the long c-axis roughly along the crystal rotation axis if possible. On 16 Aug 2013, at 16:38, Mahesh Lingaraju wrote: Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Maheshimage 1.pngimage 2.png
Re: [ccp4bb] Data processing - twinned xtals
Hi Mahesh In addition to what Mark and Juergen have written, it looks to me like you have very high mosaicity in one direction - the first image shows no distinct lunes; I suspect that may have something to do with the failure to process - but what do you mean when you say you cannot process it? Was the failure at the indexing or integration stage? If the failure is at the indexing stage, it's possible that you may make some progress in indexing if you increase the I/sig(I) threshold in Mosflm processing. Looking closely at some of the spots in the first image it does look like you have more than one lattice there, though I'm not sure I'd describe it as twinning. On 16 Aug 2013, at 15:38, Mahesh Lingaraju wrote: Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Maheshimage 1.pngimage 2.png Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] Data processing - twinned xtals
This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Data processing - twinned xtals
for #2) I'd suggest get some of those Mitigen loops that are titled. I assume you have hexagonal plates as crystals and you really want to shoot along the thin area of the crystal down the sixfold. With normal loops it's an art to get that crystal to sit upright in the loop but not impossible if you take smaller loops. My longest axis collected was 420 Å to ~2 Å resolution by this method. Jürgen On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote: This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Data processing - twinned xtals
This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Data processing - twinned xtals
This is continuation of the previous message, which was sent prematurely. In case of crystals with one, very long unit cell data collection strategy needs to be carefully chosen. Ad. 2) What would be a better way to collect data in the future? First, the detector needs to be placed far back enough, so that the spots are resolved; at minimum when the longest unit cell is in the plane of the detector (perpendicular to the beam). To satisfy this condition, it is best to rotate on the axis that is parallel (or close to parallel, within 30 degrees) to the longest unit cell. However, this can be difficult to achieve in some cases. There are two types of workaround in such a situation: a) if the crystal has low mosaicity, the spots may be resolved in angular direction, if a short oscillation is used to collect images; HKL has no problems with 0.1 degree oscillation range; b) in the case of mosaic crystals, when a) doesn't work, a partial solution is increase the detector distance. There will be still a region of reciprocal lattice where the data will be lost due to overlap, but this region may be small enough for the data to be used in structure solution. There is no indication that the particular crystal presented is twinned or highly mosaic, so chances are good that this project will be solved. Zbyszek Otwinowski Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] Data processing - twinned xtals
tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine with most SSRL beamlines except of the 12-2 microfocus - but that might have been fixed in the meantime. Jürgen On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote: for #2) I'd suggest get some of those Mitigen loops that are titled. I assume you have hexagonal plates as crystals and you really want to shoot along the thin area of the crystal down the sixfold. With normal loops it's an art to get that crystal to sit upright in the loop but not impossible if you take smaller loops. My longest axis collected was 420 Å to ~2 Å resolution by this method. Jürgen On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote: This is clearly a case of a crystal with a very long unit cell; a case which should be approached mindfully. HKL2000 has a default search for indexing solutions such that diffraction along the longest unit cell will be resolved, with the assumed spot size. The problem with such diffraction has 2 aspects: 1) how to process the already collected data where the spots are close to each other; 2) how to collect future data. Ad 1) The best solution is to reduce the spot size, so the spots are resolved. This may require an adjustment of spot size by a single pixel; one should not only change spot radius, but also change the box size between even and odd number of pixels in the box dimensions. Just changing the spot radius changes the spot diameter by an even number of pixels, so if one wants to change the spot diameter by one pixel, one has to change the box size. This is the consequence of the spot being in the center of the box. Just during indexing, there is also a workaround by specifying the command before indexing: longest vector followed by a number that defines the upper limit of the cell size. This may help finding indexing, but will create overlaps between spots during refinement and integration. This dataset presents a problem of collecting data by rotating on the axis perpendicular to the long unit cell. In consequence, the Image 1 has essentially (barely differing in centroid position) overlapping spots, so it would be hard to process them meaningfully by any program. Ad. 2) What would be a better way to collect data in the future? Hi CCP4 folks I have a data set which is looks twinned ( see the image-1 - I zoomed on to the image so that one can spot the twinning. Furthermore, the spots are very smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using HKL2000 and mosflm to process this data but i cannot process it. I was wondering if anyone has any ideas as to how to process this data or comments on whether this data is even useful. Also, I would really appreciate if someone could share their experiences on solving twinning issues during crystal growth Thanks in advance ! Mahesh[image: Inline image 2][image: Inline image 3] Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.eduhttp://lupo.jhsph.edu/ .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
