Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
Hi , Seems to me that crystal packing is not good thats why mosaic diffraction, this might be due to DNA so you can increase diffraction quality by using 1 or 2 nucleotide sticky end DNA for crystallization which can help in crystal packing . Vandna Kukshal Postdoctral Research Associate Dept. Biochemistry and Molecular Biophysics Washington University School of Medicine 660 S. Euclid, Campus Box 8231 St. Louis, MO 63110
Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
Hi Appu, nothing really to worry about if you process your data with XDS or d*trek using 3D profile fitting. You still should be able to get something useful out of this data. If you look at 90.png (I'm attaching a zoomed area of your image), you see your crystal is either split or you had two crystals in the loop. Look at the second row of reflections when starting from the beam stop position. Since the first and third row does not follow the same trend, I would think you have a second crystal in a slightly different orientation grown with the main crystal. [cid:4DB7FA0D-B1B8-44C0-A7DD-B087B0E83914@sph.ad.jhsph.edu] You should not try to index on all reflections, try getting one single lattice and ignore the other one. You will run into overlapping issues from the two lattices but you might still get good enough data to try MR when you process with XDS. With XDS you have the advantage of collecting all strong reflections over the whole dataset and then index on those. You can download it from here: http://xds.mpimf-heidelberg.mpg.de Jürgen .. 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 On Aug 1, 2013, at 9:44 AM, Appu kumar wrote: Dear all ccp4 user, We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A region but the whole data is completely spreaded to 2.8A. There are many spots which are very near to each other as if they were merged but closely placed. When we are trying to index the data, its not picking all the spots correctly and giving a unit cell dimension variable from 150 to 500A in one of the axis. Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried processing with HKL2000 but not able to index it., I am attaching four 4 images for every 90 degree frames collected. Please look at these images and give your valuable input regarding indexing problem. your suggestions and support will be highly appreciated. Please guide me and help me sorting out the problem. Thank you <90.png><180.png><250.png><371.png><0.png> <>
Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
Dear Appu, one direction of your cristal has very anistropic diffraction. I think your cristal has grown like multi-layer. The problem is probably due to the DNA. If the different "layer" of your cristal are not very well aligned, you have this type of problem. In my opinion, you can try to select a subset of your data in the best direction and try to find your cell parameters and space grp. After that, if you have high symmetry parameters it could be sufficient with the subset, or you can try to reindex the other part of your data. I collect a lot of this type of data set. And all the time it was difficult to deal with. If you have time, and if your cristal has not suffer from radiation damage, you can try smaller oscillation angle. Hope to help you Le 01/08/13 15:44, Appu kumar a écrit : Dear all ccp4 user, We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A region but the whole data is completely spreaded to 2.8A. There are many spots which are very near to each other as if they were merged but closely placed. When we are trying to index the data, its not picking all the spots correctly and giving a unit cell dimension variable from 150 to 500A in one of the axis. Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried processing with HKL2000 but not able to index it., I am attaching four 4 images for every 90 degree frames collected. Please look at these images and give your valuable input regarding indexing problem. your suggestions and support will be highly appreciated. Please guide me and help me sorting out the problem. Thank you
Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
That looks like a garden variety mosaic/kinda-crappy crystal. Without being there, it's impossible to tell if it was morphology (e.g. cracks and divots, etc.), crystal handling, or just bad luck. The bottom line is you need better data. The good news is that those crystals diffract to a reasonably good resolution on your home source. The bad news is that the data you have in hand are probably not that useful. It might be a case of just trying 50 crystals before you get a good one or you might have to modify your cryo, crystal growth, etc. Good luck. _ Eric A. Toth, Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine 108 North Greene St. Baltimore, MD 21201 Email: et...@som.umaryland.edu<mailto:etoth...@umaryland.edu> Phone: x-410-706-5345 Fax: x-410-706-8297 http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032 http://crystal.umaryland.edu<http://crystal.umaryland.edu/> [Description: Description: Description: UMSOM New logo] A Third Century Where Discovery Transforms Medicine From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Appu kumar Sent: Thursday, August 01, 2013 9:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Spots not getting indexed properly in protein-DNA complex Dear all ccp4 user, We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A region but the whole data is completely spreaded to 2.8A. There are many spots which are very near to each other as if they were merged but closely placed. When we are trying to index the data, its not picking all the spots correctly and giving a unit cell dimension variable from 150 to 500A in one of the axis. Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried processing with HKL2000 but not able to index it., I am attaching four 4 images for every 90 degree frames collected. Please look at these images and give your valuable input regarding indexing problem. your suggestions and support will be highly appreciated. Please guide me and help me sorting out the problem. Thank you <>
