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CCP4BB  March 2009

CCP4BB March 2009

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Subject:

Re: two identical proteins in one asymmetric unit

From:

Phoebe Rice <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Tue, 24 Mar 2009 15:56:58 -0500

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (1 lines)

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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