Dear Matt, I am afraid you are in a difficult position. With poor 4Å data, you need a convincing MR solution to be sure that the solution is correct. The large number of molecules in the a.s.u. is probably causing the poor diffraction.
Things I would do are: If you have more crystals, try them all. One of them may diffract better or have a better behaved crystal packing. If you don’t have more crystals, grow more and try variations and additives. It probably won’t work because your protein is monomeric, but try to construct a model of a dimer or tetramer, based on the crystal packing of suitable search models. I am afraid that this is not going to work with NMR models. Trim your model, e.g. remove potentially flexible surface loops and large side chains on the surface. Try a different construct. A slightly longer or shorter construct may give a radically different crystal packing. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthew Bratkowski Gesendet: Montag, 19. Mai 2014 22:48 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement Hello all, Thank you for you suggestions. I took a look at the crystal packing for the solution with one molecule per asu, and the next closest molecule is 50 angstroms away, suggesting that this is not likely the correct solution. I have also tried MR with a number of different molecules per asu. In some cases I get better packing, but I have not yet gotten a solution that looks to refine well. In addition to my previous information, I would like to add the following: 1. The resolution of my data is not particular great. I get ~4 A resolution at best and spots are rather weak even with almost no beam attenuation. 2. The search model that gives the best solution (in terms of contrast score in MolRep) is an NMR structure. I have heard that MR with NMR structures can possibly give false solutions. An alternate crystal structure that I tried using gave much poorer contrast socres overall, regardless of the number of molecules in the asu to search for. If anyone has any additonal suggestion, I would appreciate them. Thanks, Matt On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito <rmgarav...@gmail.com<mailto:rmgarav...@gmail.com>> wrote: Matt, In addition to the suggestions of the others, have you done a simple self rotation function? It can tell you quite a bit about how things are packed and give you strict criteria for choosing one solution over another. As Roger said, choosing an even number of monomers in the ASU is a good strategy, particularly if the self rotation function shows NCS 2-folds. Also, a calculated Matthews coefficient is NEVER correct, it is a mere guideline; it only has validity for any particularly crystal form AFTER the fact. Let the number of monomers in the ASU vary from 6-10; I have had MR cases that have had as little as 40% solvent to 70% solvent, where the calculated Matthews coefficient was quite "wrong" (i.e., the most common value observed in OTHER crystals). Two things to watch out for are: (1) An odd number of monomers in the ASU. I have had 1 1/2 dimers in an ASU (the 1/2 dimer is paired with another in a neighboring ASU). It is sometimes confusing to people and occasionally difficult to solve with some MR programs due to clashes. (2) Translation symmetry, which still can confuse some programs (but they are are getting better at detecting it). Finally, as Herman pointed out, look at the packing of any solution you are considering. It is surprising how a correct solution "looks" correct: nice intermolecular contacts and a pleasing distribution of mass throughout the unit cell (meaning expand out to at least a unit cell volume, which is easy in Pymol). Any unexplained gaps (meaning not caused by a missing domain) should be viewed critically. Regards and Good Luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724<tel:%28517%29%20355-9724> Lab: (517) 353-9125<tel:%28517%29%20353-9125> FAX: (517) 353-9334<tel:%28517%29%20353-9334> Email: rmgarav...@gmail.com<mailto:garav...@gmail.com> **************************************************************** On May 15, 2014, at 6:50 PM, Matthew Bratkowski <mab...@cornell.edu<mailto:mab...@cornell.edu>> wrote: Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any suggestions would be helpful. Thanks, Matt
