CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> wrote on 05/22/2008 10:47:24 AM:
> Dear All > > I am processing data from a crystal for a large macromolecular complex > with mosflm. Cell dimensions are around 120 x 150 x 650, with a p222 > spacegroup. To avoid overlaps, we have collected data with a oscillation > of 0.1 degrees. > > When I try to process the data with mosflm, mosaicity decreases along the > processing to a very low values (0.05 to 0.1 depending on the images). The > result is that I miss a lot of spots from the images. > > I would appreciate any help regarding : > > 1.- What's is the best strategy to process such a dataset ? > 2.- Which are the critical parameters in mosflm to avoid these problems, > and how to modify them ? > 3.- and finally, can I use this data or your advice is to try to get > crystals in a different spacegroup ? > > Many thanks > > Best regards > > Francisco > > Hi Francisco - I've got no good advice for processing this data; my suggestions are for the next data collection to help minimize the overlap problem. First, get an X-ray beam with the smallest diameter and smallest divergence you can. This means a synchrotron, of course. If the beamline you're at allows refocusing of the beam to minimize the spot size at the detector, take advantage of this. Next, get the largest detector you can, because you're going to be pushing it waaaay back. A 2k x 2k CCD won't work here - look for 3x3 or even a big image plate detector. The number of pixels is less important than the physical surface area. When collecting, move the detector as far back as possible, using 2-theta offset to keep the high resolution completeness as high as you can. You may need to do some fairly complicated data collection strategies to get a complete dataset. You need to have the long unit cell axis roughly (but not perfectly) aligned with the rotation axis of the goniometer. Try for about a 20 degree offset - if it's perfectly aligned, you'll have an uncollected region along c*. If you have a kappa goniometer at your beamline, this is not too bad, although it's much much easier if you can look at the crystal and know where the long axis is pointing. Trying to do this by inspecting test diffraction images is very difficult and makes my head hurt. If you only have a single axis goniometer at your beamline, all is not lost. Buy one of these: http://www.hamptonresearch.com/products/ProductDetails.aspx?cid=25&sid=151&pid=414 and you now have a crude, manually adjustable kappa: you swing the arc out for kappa angle, and spin the crystal pin on its magnetic base for phi rotation. Practice this at home first!! Using small rotation angles helps, but only up to a point. If you have 0.6 degree mosaicity, then a 1 degree oscillation gives you 1.6 degrees worth of data on the image. A 0.5 degree oscillation reduces this to 1.1 degrees of data, but a 0.1 degree oscillation is still 0.7 deg of data, and five times the number of images! I wouldn't go below 50% of the crystal mosaicity for my oscillation angle. Hope that's useful to you. - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
