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On Tuesday 24 March 2009 12:20:17 Mischa Machius wrote: > 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. The problem can be mitigated enormously by parameterizing the NCS restraints in terms of torsion angles rather than coordinate transforms. This is what shelx does, and it works a fair treat. The point is that is this representation the deviation from NCS caused by an ideal hinge motion shows up only in the handful of torsions corresponding to the hinge point. This means that atoms remote from the hinge point can still adhere closely to the NCS restraint even though there is no single NCS operation that will superimpose them. Ethan > > 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: [log in to unmask] > > > > 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: [log in to unmask] > >> Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa > >> ******************************************************** > >> > >> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf > >> Of Jim Fairman > >> Sent: Tuesday, March 24, 2009 11:25 AM > >> To: [log in to unmask] > >> 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 > >> <[log in to unmask]> 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 <[log in to unmask]>: > >> > 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 [log in to unmask] [log in to unmask] > > -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742