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Apologies for continuing to top-post here... If the cleavage would only remove 28 amino acids from a 500 amino acid chain, it could easily be missed by SDS-PAGE, although mass spec would catch it. However, I don't think you can get enough sample from a dissolved crystal to run mass spec. To the original poster: you need to describe the electron density break to us in more detail, or better yet, direct us to some screenshots. What is your resolution? How good is the electron density in the vicinity of the break? Can you see the side chains of residues 28 and 29? A true peptide bond cleavage will leave free amino and carboxy termini which will not fit in the same space as a peptide bond - even at modest resolution, you should be able to see that C-alpha #28 and C-alpha #29 are not the canonical 3.8 A apart; at high resolution, you would hopefully see the extra oxygen of the COOH terminus. - Matt -- Matthew Franklin phone:(917)606-4116 Senior Scientist, ImClone Systems fax:(212)645-2054 180 Varick Street, 6th floor New York, NY 10014 [EMAIL PROTECTED] wrote on 12/01/2005 11:59:14 AM: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Short answer - CAN'T BE. > > If you dissolve crystals and run SDS PAGE and/or mass-spec and do not see the > cleavage it means that there is an error in your electron density. Some > disorder is really hard to model even having the atomic resolution data, and > some main chain bonds might have much lower density than the others creating > an appearance of cleavage. > > Petr > > On Thursday 01 December 2005 02:01 am, Jinkwang wrote: > > Hi all, > > Routine MR solution of a mutant structure was an easy task until I found a > > clear cleavage within the polypeptide chain. This cleavage is reproducible > > only in crystal structure, and biochemical studies such as SDS-PAGE, > > N-terminal sequencing or Mass spectra never indicate any cleavage in the > > chain. > > > > > > > > The protein has about 700 amino acids, divided into alpha (200 a.a) and > > beta (500) chains. The cleavage is found at 28-29 of the beta chain in the > > crystal. Wondering whether this could occur only in crystal states, the > > crystals were dissolved, but the biochemical studies did not show any > > cleavage, but crystal structure once again shows. > > > > > > > > Any comments are highly appreciated. > > > > > > > > Regards, > > > > Jin Kwang 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.
