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At the risk of going over previously covered ground... You *should* be able to get Mass-Spec data from a crystal. Our Mass-Spec guys here routinely cut bands out of stained SDS-PAGE gels and get accurate enough peptide masses to ID unknown proteins... Loss of 28 residues should easily be detectable. On 1/12/05 9:43 pm, "[EMAIL PROTECTED]" <[EMAIL PROTECTED]> thought: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > 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. > > -- *************************************************************** Dr David Briggs | Structural Biology Lab (309) | Tel : (+44)(0)20 7269 3360 Cancer Research UK | 44 Lincoln's Inn Fields | Holborn | London | UK | WC2A 3PX | ****************************************************************
