Ian, I know that sometimes Fo(complex)-Fo(apo) cannot be done because of nonisomorphism. We've had a lot of success with this with the dioxygenases because there is no large scale alteration in the active site. As for the technique itself, Brian Matthews drilled this into me when I was a postdoc in his lab.
All the best, Doug Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology & Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -----Original Message----- From: Ian Tickle [mailto:[email protected]] Sent: Wednesday, June 17, 2009 12:08 PM To: Doug Ohlendorf Cc: [email protected] Subject: RE: [ccp4bb] Difference Map images Hi Douglas Do you have some references with examples of this technique? In my experience this is a difficult experiment to perform routinely except in a few special cases. The first problem is that soaking the ligand can easily induce significant cell dimension changes, which if large enough causes non-isomorphism errors to wipe out the advantage of the similarity of apo & complex crystal. One may be able to get around this of course by soaking the apo crystal in the same concentration of DMSO (or whatever solvent you use) as the complex was soaked in, but even then if the ligand is a tight binder it can induce conformational changes that again cause significant non-isomorphism errors. Then even if you can get around these problems, you have the problem that freezing the crystals usually causes differential cell dimension changes, possibly due to differing concentrations of organic solvent, but more likely it's simply that it's almost impossible to control the rate of freezing reproducibly. Is there a trick to avoid this? - of course you could simply not freeze, but then this would limit it to strongly diffracting crystals where you could afford to attenuate the beam to reduce radiation damage to an acceptable level. Cheers -- Ian > -----Original Message----- > From: [email protected] [mailto:[email protected]] On > Behalf Of Doug Ohlendorf > Sent: 17 June 2009 16:41 > To: [email protected] > Subject: RE: [ccp4bb] Difference Map images > > Andy, > > One important thing if your complex crystals are isomorphous with apo is > to > use nFo(complex) - Fo(apo). These maps give the maximum information as the > uninterpretable 'stuff' in the Fo-Fc map is likely quite similar in both > crystal forms so the difference signal should be cleaner. > > > Douglas H. Ohlendorf Phone: > 612-624-8436 > Professor FAX: > 612-624-5121 > Dept. of Biochemistry, Molecular Biology & Biophysics > Twin Cities Campus, University of Minnesota > Lab web site: > http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html > > > -----Original Message----- > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of ANDY > DODDS > Sent: Wednesday, June 17, 2009 8:30 AM > To: [email protected] > Subject: [ccp4bb] Difference Map images > > Hello, > > I was wondering what people used to generate difference map images of, > say, a ligand in their structures? > > e.g. Figure 2a here > > http://journals.iucr.org/f/issues/2009/05/00/tt5012/tt5012.pdf > > > > > > Cheers, > > Andy Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [email protected] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
