 |
 |
Hi Sylvia: > -----Original Message----- > From: Sylvia Fanucchi [mailto:[log in to unmask]] > Sent: 19 October 2009 15:06 > To: Ian Tickle > Subject: RE: how to improve Rfree? > > Hi Ian > > Yes, sorry, admittedly this is the first time I'm doing this and I'm not > sure of all the checks that need to be made. I have been checking the > validity of my model in coot. > > I've been under the impression that the initial factor that will tell > you whether the refinement has been any good and whether the model is on > the right track is the R-factor. I figured that once I'd got that to a > satisfactory number I would run the model through validating programmes > such as procheck or molprobity. Am I missing something fundamental? This > would not surprise me since I am very new to this and have essentially > taught myself most of everything I know. Procheck/Molprobity is a good start for geometric validation! It's also important to look for obvious unexplained peaks in the difference Fourier since good agreement of the model with the X-ray data is also important (Coot will help with this); and there are programs which list the real-space density correlation coefficients and/or density RMSD that may help identify problem areas. The refinement program often also has tables and/or graphs of other useful statistics such as structure factor correlation coefficients and log-likelihood, as well as R factors of course. > My resolution is 1.6A although I have cut it to 1.8A to bring the > R-factor down. I've been performing restrained refinement in refmac5 > using the default settings. The solvent content is 40% An Rfree-R difference of 0.08 at 1.8 Ang does indeed seem somewhat high, though the low solvent content may go some way towards explaining it (since it means you will have fewer data in comparison with other structures of similar size and resolution). In addition if your overall completeness is say < 0.95 (or < 0.8 in the outer shell) that won't help. However I assume you mean Rmerge not Rwork: the purpose of refinement is not to reduce the R factor but to get the model which best explains the observations (including the restraints), and also makes sense in comparison with the body of other structures already determined: rejecting data on the basis of Rwork and/or Rfree may not be the best way to achieve this! Personally I reject data based only on shell completeness (> 0.8) and mean I/sd(I) (> 1.5); I don't use Rmerge for this - see recent BB discussions. > Someone told me once that if the Rfree increased with successive rounds > of refinement while the R-factor decreased, something was very wrong and > that was why I was concerned. If Rfree (or -LLfree) *ends up* being higher with one refined model / parameterisation / weighting scheme compared with another (with the same data), then one would reject the first model. However what you can't do is compare the models on the basis of the intermediate Rfree/LLfree values. Cheers -- Ian Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [log in to unmask] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674