Re: [ccp4bb] two identical proteins in one asymmetric unit

[Roger Rowlett]

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:CCP4BB@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