CCP4 bulletin board <[email protected]> wrote on 05/14/2009 03:42:05 PM:
> Dear Crystallographers, > > I have exactly two spherulite crystals of a protein-peptide complex which > have a fluorescently-labelled peptide in them, and are therefore nicely > colorful in both the light and fluorescence microscopes, making it easier to > know that at least the peptide is in the crystal. However, they are not > reproducible. Having gone through the usual list of possible variations > which might account for the irreproducibility, I have hit the bottom of the > barrel. I was thinking that since the original crystals grew in utter > darkness, undisturbed for two weeks while I was away, they were able to > nucleate. Is it possible that light exciting the fluorophores is detrimental > to crystallization? Or perhaps the complete uniformity of temperature? Even > microseeding from one of the spherulites produced nothing (except in the > original well.) Any brilliant suggestions welcome... > > Jacob Keller > Hi Jacob - I just want to raise a warning, since a very similar situation bit me back in grad school. I was trying to crystallize RecA with a fluorescently-labeled oligo, and just like you, I got green-glowing crystals, along with some glowing precipitate. I got extremely excited, and a couple of months later had the structure done. There was no DNA in the structure. I had crystallized unliganded RecA, and the fluorescent DNA had precipitated all over the outside of the crystal, painting it and making it look green! Are you certain that your peptide doesn't precipitate under your crystallization conditions? I hope for your sake that my problem is not yours... - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly & Company 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.
