Hi Bill Scott used a RNA fragment based MR approach in his paper on Ribozyme structure.
The Structural Basis of Ribozyme-Catalyzed RNA Assembly Michael P. Robertson and William G. Scott Science 16 March 2007: Vol. 315. no. 5818, pp. 1549 - 1553 In our lab we had an interesting time with the two forms of Glutamate Decarboxylase. This is probably not as extreme as some of the stories posted, but may be useful. Initially we crystallised GAD65, at 2.3A resolution with 1 Mol in the ASU and C2221 with the two molecules in the dimer related by crystallographic symmetry. The MR probe (Dopa Decarboxylase) was about 17% identical and comprised ~33% of the final GAD structure (essentially part of the PLP domain). While we were able to get a clear MR solution using PHASER (correctness confirmed by the packing of the crystallographic dimer, which is physiologically relevant), we were unable to get over model bias. To address this we first tried heavy atom soaks to attempt phased MR, but always ran into serious non-isomorphous issues with the crystals. Since GAD has to be produced in yeast, MAD was not really an option. As a last resort, we crystallised GAD67 (~75% identical to GAD65) again crystals diffracted to 2.3A resolution but this time SG P21 with two molecules in the dimer related by NCS. This time, using a similar MR probe, we again got a clear solution, and in refinement NCS averaging won the day and we were to complete the structure. Its worth noting that we had a pretty unpleasant time trying a LOT of different MR models (at least 15-20) even for GAD67 and we had to progress very carefully with the building. Once GAD67 was solved, however, phasing, building and refinement of GAD65 was straightforward (as expected), using a GAD67 monomer as an MR probe. The work is described in: Fenalti et al GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat Struct Mol Biol. 2007 Apr;14(4):280-6. Epub 2007 Mar 25 In the end, we were happy since comparison of the two structures was what we wanted to do. However, if only we had gone for GAD67 first, we would have saved a lot of pain!!! Cheers James "Bryan W. Lepore" <[EMAIL PROTECTED]> wrote:> > a quick summary on 'the worst MR probes' > > Pierre Rizkallah: > X-ray Structure Solution of Amaryllis Lectin by Molecular Replacement > with Only 4% of the Total Diffracting Matter. > L. Chantalat, S.D. Wood, P.J. Rizkallah, C.D. Reynolds (1996). > Acta Crystallographica, D52, pp. 1146-1152 > > Poul Nissen: > Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L, Clark BF, > Nyborg J (1995). "Crystal structure of the ternary complex of > Phe-tRNAPhe, > EF-Tu, and a GTP analog." Science, 270, 1464-72. > > Anderson, et. al. (from Manfred Weiss) acta cryst d 52, 469-480 > > other pubs worth checking out: > > Schwarzenbacher, et. al. acta cryst d 60 1229 2004 > Adams, et. al. acta cryst d 55, 181, 1999 > all phaser refs. > > i also got a report of the correct phaser solution with > Z-score=5.2/LLG=49 > - for the uninitiated, a phaser Z-score of 5 to 6 is an 'unlikely' > solution. > > any other references, stats appreciated > > thanks. > > -bryan -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
