My favorite trick was to define domain-wise ncs restraints, extensively minimize with and without them, then plot the difference in real-space R factor per residue. Ones that jump up when restrained are usually involved in crystal packing, etc, and should be removed from the restraints. In my clumsy hands, this required 4 cns scripts followed by importing the real-space R lists into excel. There must be a better way? Phoebe
---- Original message ---- >Date: Tue, 24 Mar 2009 14:20:17 -0500 >From: Mischa Machius <mischa.mach...@utsouthwestern.edu> >Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit >To: CCP4BB@JISCMAIL.AC.UK > > Having dealt with quite a few cases of more than one > molecule in the AU (including a couple of dreaded > 12-meric assemblies... bleah), I am still looking > for the best way to identify proper NCS operators > for the myriad of potential combinations of > fragments. > As has been said, it is generally worthwhile to > identify the equivalent portions of the molecules > and appropriate NCS weights, not only > for potentially finding something interesting in > terms of biology, but also for doing the best > possible refinement job. > I therefore wish there were better tools for this > purpose. Overall, I think this area has not received > proper attention yet. But it should, because I have > the feeling that the impact of a great set of tools > would be immense. > Eternally hopeful - MM > -------------------------------------------------------------------------------- > Mischa Machius, PhD > Associate Professor > Department of Biochemistry > UT Southwestern Medical Center at Dallas > 5323 Harry Hines Blvd.; ND10.214A > Dallas, TX 75390-8816; U.S.A. > Tel: +1 214 645 6381 > Fax: +1 214 645 6353 > On Mar 24, 2009, at 1:22 PM, Roger Rowlett wrote: > > I had a student solve a medium resolution (2.3 A) > data set with (unfortunately) 12 identical protein > chains in the asymmetric unit. To save a little > time, and to take advantage of a large amount of > potential averaging we used NCS to do the initial > phase of the refinement. For 10 of the 12 chains, > everything was hunky-dory. For the 11th and 12th > chains, however, there was an extremely messy area > of high-sigma difference map density that turned > out to be a very interesting ligand-binding > interaction. Releasing the symmetry constraints > resulted in a very sharp map of the protein chain > rearrangement and bound ligand in the two > "different" chains. > > In general, even with homodimers and homotetramers > in the ASU, we find that there are often subtle > but significant differences in individual protein > chains, especially around packing contacts and > external loops of the protein. > > Cheers, > > -- > > ------------------------------------------------ > > Roger S. Rowlett > Professor > Colgate University Presidential Scholar > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@mail.colgate.edu > > Skrzypczak-Jankun, Ewa wrote: > > I have seen proteins refined as ‘the same’, > modeled to an averaged map etc only to have one > of them with much higher Bj because most likely > they are NOT the same so watch out by treating > them as ‘the same’ you are losing the very > valuable information that you might be looking > for > > Ewa > > > > ******************************************************** > > Dr Ewa Skrzypczak-Jankun > Associate Professor > > University of Toledo > Office: Dowling Hall > r.2257 > > Health Science Campus > Phone: 419-383-5414 > > Urology Department Mail Stop #1091 > Fax: 419-383-3785 > > 3000 Arlington Ave. > e-mail: > ewa.skrzypczak-jan...@utoledo.edu > > Toledo OH 43614-2598 > web: > http://golemxiv.dh.meduohio.edu/~ewa > > ******************************************************** > > > > ---------------------------------------------------- > > From: CCP4 bulletin board > [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim > Fairman > Sent: Tuesday, March 24, 2009 11:25 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] two identical proteins in > one asymmetric unit > > > > Sang Hoon, > > Each molecule in the asymmetric unit is most > likely different. I work on a protein that > crystallizes as a homodimer with 2 molecules per > asymmetric unit and there are some differences > between the two (eg: electron density visible > for the 14 N-terminal residues in one molecule, > but not the other). > > Cheers, Jim > > On Tue, Mar 24, 2009 at 11:03 AM, Folmer > Fredslund <folm...@gmail.com> wrote: > > Dear Sang > > They are really different! > > And I guess you would probably want to use NCS > restraints depending on > your resolution. > > Regards, > Folmer > > 2009/3/24 Sang Hoon Joo <s...@duke.edu>: > > I am refining my crystal structure in which I > have two identical > > chains in one asymmetric unit. > > Space group is H32 and each chain yields me a > biological trimer as expected. > > The problem is, do I have to assume they are > identical, or they are > > really different. > > After each cycle of refinement, if I try to > align two molecules I get > > ~ 0.17 RMSD. > > -- > > Sang Hoon Joo, PhD > > Postdoctoral Associate > > Duke University > > 239 Nanaline H. Duke > > Box 3711, DUMC > > Durham, NC 27710 > > > > -- > Jim Fairman > Graduate Research Assistant > Department of Biochemistry, Cellular, and > Molecular Biology (BCMB) > University of Tennessee -- Knoxville > 216-368-3337 jfair...@utk.edu > james.fair...@case.edu Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
