Re: [ccp4bb] and another MR problem

[Roger Rowlett]

When doing MR, I usually try Phaser and EPMR. Phaser rarely fails for a high-homology MR search, but can have difficulty fitting 3 or more protein units in the ASU. EPMR is a little better, in my experience, in ferreting out a solution for 3 or more protein units. If you are running the Matthews Probability Calculator in Phaser (I think it is called Cell Content Analysis) you should take that result with a grain of salt, and consider additional solutions, e.g., 2 and 4 chains if the suggestion is 3 chains per ASU. While most proteins fall within the expected range of solvent content, it is possible to have solutions on the fringes. (We just had one recently with 67% solvent content.) In general, searching with larger protein units is better than with monomers, if you know the structure of the multimers. That is, searching with dimers instead of monomers is usually more effective, especially if this will reduce the number of protein units to be placed to 3 or fewer. If the biological unit is a tetramer, sometimes you can get a good solution with AB dimers but not with BC, CD, or AD dimers, etc. If running Phaser, be sure to allow for extra clashes in your search, or you may reject all of the possible solutions. It is not unreasonable to allow for 30 or more clashes in your initial trials. Also, you may want to retain 65% the best rotation peaks instead of the default 75%, to improve your chances of finding a solution. If Phaser doesn't work, try EPMR. I have not had it fail yet for an MR search for 3 or fewer protein chains per ASU, and high-homology search models. The success of EPMR can be improved by including a little more of the high-resolution data, although this will slow things down. In a difficult case, we extended the data used by EPMR almost to the diffraction limit of the crystal to get a good MR solution. The good news is that C2 is a relatively simple search space: no alternative space groups or screw axis combinations, or reindexing of data required. If you suspect 2 or 3 protein chains in the ASU, try for a partial solution for 1 or 2 chains, then examine the result for packing, which often gives you some clues as to where and how many additional chains might be placed. Cheers, and good luck. On 8/23/2010 12:10 PM, Teresa De la Mora wrote: Dear all I have a molecular replacement problem and I really need your help with this one. My protein crystallized in C2 instead of P21 like it did before. Now when I tried to do MR to place it in C2 it doesn't work, I get no solution. This protein has been crystallized in P212121, P31, P43 and C2 and it has two loops in different regions that depending on the packing, they'll show density so it is flexible. When I crystallized it the first time in P21 and used a publish model I had no problem at all to place it, just right now. I've have tried molrep, phaser and phenix with different resolution ranges (40-3.0, 20-3.0, 10-3.0) and different models, i.e. publish structures or the one I got at P21. I also tried doing rotation first at different resolutions then compare the peaks obtained and picked the ones that are consistent and give that for translation search and yet no solution. The resolution is 2.4 A and the Matthews coefficient predicts 3 molecules in ASU with 43.6% solvent. I also tried searching for 2 molecules just in case that it is high solvent content (62.5%) as monomer and as dimer and yet no solution. The data itself processed well and I've processed in HKL and d*trek and used both processed files and yet no solution. Completeness is 96.9% overall and 82% (2.49-2.40) shell. Redundancy is 3.5 overall and 2.6 at the(2.49-2.40) shell. Here is the table from HKL:  Lower   Upper  Average  Average     Norm. Linear Square      Angstrom              I       error    stat.  Chi**2  R-fac  R-fac       50.00   5.17   901.9    35.9    10.2  1.105  0.041  0.051         5.17   4.10   962.8    47.7    11.6  0.971  0.045  0.049         4.10   3.58   496.4    25.9     9.2  1.194  0.063  0.067         3.58   3.26   309.9    15.2     8.4  1.360  0.069  0.067         3.26   3.02   153.9      9.9     7.2  1.456  0.105  0.098         3.02   2.85   100.8      7.3     6.9  1.864  0.141  0.120         2.85   2.70    57.4       7.0     6.8  1.584  0.200  0.162         2.70   2.59    41.9       7.5     7.0  1.348  0.246  0.220         2.59   2.49    30.5       7.4     7.3  1.526  0.329  0.285         2.49   2.40    22.5       8.0     7.8  1.347  0.388  0.361   All reflections    319.9    17.6     8.3  1.366  0.065  0.056 Would you please, please give me some tips, tricks, advice, encouragement? :-)  Thank you so much  Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry  University of Minnesota  8-101 Weaver-Densford Hall  308 Harvard St. SE, Minneapolis, MN 55455  Lab phone (612) 626-5226  "If you never did you should. These things are fun and fun is good" Dr. Seuss -- Roger S. Rowlett Professor 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...@colgate.edu