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 <[log in to unmask]>
>Subject: Re: [ccp4bb] two identical proteins in one
asymmetric unit
>To: [log in to unmask]
>
> 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: [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]
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
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