Re: [ccp4bb] worst mr probes
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)
Re: [ccp4bb] Solvent content of membrane protein crystals
I would use a very general definition for "solvent", including disordered detergent and lipids. As you know in many cases ordered detergents and lipids have been modeled in the coordinates, so they are part of the model not the solvent. In some cases I think waters should be included in the model not solvent- say for structural waters buried in the protein at least. Ed Savvas Savvides wrote: Dear colleagues, in estimating the solvent content of membrane protein crystals it would only seem reasonable that micelle size should also be taken into account. Depending on the aggregation number and MW of a given detergent, the concentation of detergent used, and the buffer conditions, one may have micelles on the order of 15-25 kDa or even 35-50 kDa for detergents with alkyl chains of more than 10 carbons. However, when I took a look in a handful of papers reporting Matthews' numbers for membrane protein crystals, it became apparent that only the protein MW is used in such estimates. I am beginning to wonder if one should even bother reporting a Matthews number for a membrane protein crystal given the uncertainties surrounding size and role of micelles in crystal packing. Any thoughts on this? best wishes Savvas
[ccp4bb] Solvent content of membrane protein crystals
Dear colleagues, in estimating the solvent content of membrane protein crystals it would only seem reasonable that micelle size should also be taken into account. Depending on the aggregation number and MW of a given detergent, the concentation of detergent used, and the buffer conditions, one may have micelles on the order of 15-25 kDa or even 35-50 kDa for detergents with alkyl chains of more than 10 carbons. However, when I took a look in a handful of papers reporting Matthews' numbers for membrane protein crystals, it became apparent that only the protein MW is used in such estimates. I am beginning to wonder if one should even bother reporting a Matthews number for a membrane protein crystal given the uncertainties surrounding size and role of micelles in crystal packing. Any thoughts on this? best wishes Savvas
Re: [ccp4bb] the worst molecular replacment probes
The paper below can put things in perspective a little bit. Bernstein and Hol (1997) Acta Crystallogr D Biol Crystallogr. 53(Pt 6):756-64. Probing the limits of the molecular replacement method: the case of Trypanosoma brucei phosphoglycerate kinase. best wishes Savvas
Re: [ccp4bb] Crystals to go
I am pretty sure the work you are referring to is R. B. G. Ravelli et. al. JSR 2007 http://dx.doi.org/10.1107/S0909049506043111 the article is open access, so anyone anywhere can click on the above link and read it. Basically, they used EM specimen preparation methodologies to embed a lysozyme crystal in plastic. The radiation damage rate was greatly reduced, but it was not zero. Recently, I have begun to think that inorganic crystals would be a good choice for a room-temperature diffraction standard, since they are more rad-hard than organics. The largest unit cell I have heard of for an inorganic is Mo2P4O15 with 441 atoms in the ASU http://dx.doi.org/10.1039/b408413f Don't know much more about it than that however, since I can't access this article. -James Holton MAD Scientist Winter, G (Graeme) wrote: Hi All, I heard about a way of preparing crystals - presented at the BSR recently as "Crystals to go" - which allows them to be carried at room temperature then cooled for data collection i.e. for beamline testing. Please could someone point me in the right direction for the instructions on how to do this, as I didn't get a chance to see the poster! Thanks, Graeme
[ccp4bb] worst mr probes summary
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
[ccp4bb] refmac and ramachandran troubles
Hi all, along the lines of a recent discussion: At the final stages of the refinement of a structure which a whole bunch of ncs (2x4chains) at 2.2A resolution in P1, I run into the following very annoying and persistent problem. The Ramachandran plot shows a whole bunch of ugly outliers. While some of them appear to be "true", ie. the same outliers in all chains w/o application of ncs, loop region, H-bonds that make sense, ..., there are a number of "misbehaving" outliers: After reciprocal space refinement some residues violate the Ramachandran plot for one or two chains only. Both 2FoFc and FoFc density clearly show that the main chain is supposed to be at a slightly different position. Real space refinement in coot puts the mainchain nicely in place on top of the ncs mates' position. Refmac then pulls things back into the forbidden region. For an illustration see here: http://picasaweb.google.com/Jan.Abendroth/RefmacAndRamachandran/ I have tried to tie down the violators with tight ncs restrains - no success. I am running refmac 5.3.0037, with TLS refinement. Rfactors seem really decent (20%/25%) as does geometry. Refmac complains about "MAKE_U_POSITIVE" problems. Any ideas? Cheers Jan -- Jan Abendroth deCode Biostructures Seattle/Bainbridge Island, WA, USA work: [EMAIL PROTECTED] home: [EMAIL PROTECTED]
Re: [ccp4bb] the worst molecular replacment probes - three months
Sorry - didn't see this - time spent on MR solution of the Phe-tRNA:EF-Tu:GDPNP structure was about three months (but this was 1994 with slow computers, two trips per year to the synchrotron etc.) Poul > To avoid excessive excitement potentially caused by such a list, people > should also indicate the time spent with a model just good enough to > initially justify the eventually futile effort. > > > Andreas > > > On 9/21/07, Bryan W. Lepore <[EMAIL PROTECTED]> wrote: >> >> would anyone be willing to share stories of the worst molecular >> replacement search probes they used to get the correct solution purely >> with MR? >> >> perhaps in terms of %-scattering, RMSD, Z-score, LLG, or other possibly >> specific scoring values. >> >> -bryan >> > -- Poul Nissen, professor, ph.d. Centre for Membrane Pumps in Cells and Disease - PUMPKIN University of Aarhus, Dept. Molecular Biology Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark http://www.mb.au.dk http://www.bioxray.dk http://www.pumpkin.au.dk http://person.au.dk/da/[EMAIL PROTECTED] Tel. +45 8942 5025, Fax +45 8612 3178, [EMAIL PROTECTED]
Re: [ccp4bb] the worst molecular replacment probes
An old example: The Phe-tRNA:EF-Tu:GDPNP ternary complex - three complexes per asymmetric unit, C2 space group, degenerate three-fold NCS (three different tRNA conformations in the trimer). 40 - 2.8 Å resolution native data (about 78% complete...) used for MR. Straight-forward placement of three EF-Tu copies using 10 - 3.5 Å resolution data (AMORE). A single copy of a truncated tRNA model (representing about 7% of the macromolecular mass of the asymmetric unit) was placed using data in the 15 - 6 Å range (AMORE) and identified by significantly better statistics of the corresponding TF solution (with EF-Tu molecules placed as prior models) compared to all other RF solutions - ~48% R-factor compared to ~52%+ for other solutions as I recall, and similar distinction of PC value. Optimisation of the RF peak value for this single solution (by variations of RF search parameters) produced a second solution in the RF top-100 peak list that also yielded a distinct solution in TF. Third tRNA molecule was placed by screening of the degenerate three-fold rotation by "manual" MR. Only low resolution data worked for the tRNA MR. Gradual model refinement (TNT) completed the structure (PDB 1TTT) and bogus occupancy refinement of tRNA complemented B-factor refinement as judged by a significantly improved Rfree value. Post-refinement using CNS with proper ML target and anisotropic scaling makes the bogus occupancy refinement obsolete - today TLS parameterisation would probably do an even better job). I didn't check but the tRNA RMSD (truncated model vs. refined structure) will probably exceed 1 Å. All pretty well decribed in: 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. Poul > would anyone be willing to share stories of the worst molecular > replacement search probes they used to get the correct solution purely > with MR? > > perhaps in terms of %-scattering, RMSD, Z-score, LLG, or other possibly > specific scoring values. > > -bryan > > -- Poul Nissen, professor, ph.d. Centre for Membrane Pumps in Cells and Disease - PUMPKIN University of Aarhus, Dept. Molecular Biology Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark http://www.mb.au.dk http://www.bioxray.dk http://www.pumpkin.au.dk http://person.au.dk/da/[EMAIL PROTECTED] Tel. +45 8942 5025, Fax +45 8612 3178, [EMAIL PROTECTED]
