CCP4 bulletin board <[email protected]> wrote on 05/10/2007 09:31:59 PM:
> Hello, > I'm an undergraduate and recently crystallized and obtained 2.9A > diffraction data for a protein which is predicted to fold into a WD40 > 7-bladed beta-propeller structure (which has been crudely verified by > cryo-EM by another lab). The space group appears to be I4(1) with > unit cell parameters 118.936 118.936 85.456 90.000 > 90.000 90.000. Using a number of different search models (which I > trimmed and aligned to my protein's sequence using Chainsaw) I have > obtained a number of MR solutions placing 2 molecules in the AU with > Phaser with high Z-scores (ranging from Z=9 to 12) that seem to pack > together nicely, so I was hoping to use this technique to solve my > structure. However, the initial Rfree for my best solution is > relatively high (0.49) and all attempts to refine the structure > result in the Rfree blowing up almost immediately. This makes me > worry that the maps I'm generating may be too model-biased to use to > generate a solution. I've tried using Prime and Switch to remove > model bias but the resulting map looks worse than the starting map. > As the predicted structure possesses so much radial symmetry (7- > fold), I'm worried that my MR solutions will never be oriented > correctly enough for me to be able to build a model. If anyone has > any suggestions for tackling this kind of molecular replacement woe, > I would greatly appreciate it. Otherwise I guess I'll just plan to > collect experimental phasing information sometime in the near future. > > I'm not sure if this is the right place to be asking this question, > perhaps you guys could direct me elsewhere. > > Thanks! > -Scott Hi Scott - You can probably overcome the 7-fold symmetry problem manually. I suggest the following: - Take a few of your search models that give the best solutions (are all the solutions roughly superimposed?) - Generate copies of the search model in each of the 7 orientations corresponding to the 7-fold symmetry. You might be able to generate a symmetry operator to do this, but I'd probably just do it by eye using O or something similar. - Submit the rotated models to optimization using Phaser. You'll use the "Refinement and phasing" option in the GUI, or the "MODE MR_RNP" keyword in the script file. You'll feed Phaser a fake solution file (the .sol file) with all Euler angles and fractional translations set to zero. - Compare the resulting LLGs of the seven symmetry-related models. With luck, one of them will stand out from the others. (Z-scores may not be meaningful here - I forget whether Phaser calculates Z-score using a random set of orientations, or just takes the mean and std. deviation of the ensemble of solutions.) - Take the best orientation (or all of them if one doesn't stand out) and try Refmac refinement. Again, one will hopefully look better than the others. Hope this works for you - it's certainly something to try while you're trying to get derivatives... And feel free to contact me if this wasn't clear enough for you to try it. - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
