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Alun, There's only any point doing it if they're truly isomorphous (e.g. by the Crick-Magdoff test), otherwise pairs of reflections with equal indices won't sample the same points in the transforms, so you could still end up with contamination. Slow cooling or pseudo-MD such as randomly shifting co-ordinates actually moves you away from convergence, and as I said convergence is where you need to be for Rfree to be meaningful and to completely erase 'memories' in the structure. Also it's a progressive thing, the closer you get to convergence, the more meaningful Rfree becomes and the more the memories are erased. Of course convergence is very elusive, just because the parameter shifts appear to be getting smaller doesn't guarantee convergence (e.g. the series 1+1/2+1/3+1/4+1/5+... diverges to infinity!), so by all means try slow-cooling, just be prepared to carefully evaluate the results. Cheers -- Ian > -----Original Message----- > From: [log in to unmask] > [mailto:[log in to unmask]] On Behalf Of Alun R. Coker > Sent: 19 December 2008 10:37 > To: [log in to unmask] > Cc: CCP4 bulletin board > Subject: Re: [ccp4bb] Transferring a Free R set. > > Hi Ian, Gerard, > > Thanks for your replies. > > Perhaps my hypothetical example wasn't a good one and may > have clouded > the issue. Could I just confirm whether or not you think its > necessary > to transfer initial free R assignment to any new data sets or to > isomorphous data sets such as substrate complexes. > > Thanks, > > Alun. > > Ian Tickle wrote: > > Hi Alun > > > > I've never seen a soaked ligand-bound structure that's > truly isomorphous > > with the native - simply soaking & freezing the crystal is virtually > > guaranteed to introduce very serious non-isomorphism, e.g. up to 5% > > change in one or more cell parameters. At least, we always > have a MR > > step to solve the structure, as rigid-body refinement alone > often fails > > to pull it in, so the R-factors always start high (> 0.4) > anyway. This > > renders the concept of a common test set totally > meaningless, since the > > native & ligand-bound reflections with the same indices > (except maybe > > the low res ones) will not be in equivalent positions in reciprocal > > space, and in any case once it has been refined down & been > through a > > few rounds of rebuilding any bias will very quickly disappear. > > > > Note that Rfree is a meaningful statistic only at convergence at the > > likelihood maximum, and at convergence all previous > 'memories' of the > > structure are erased: bias due to 'contamination' of the > test set only > > occurs as a result of failure to ensure convergence. Also I don't > > believe that any of the cases of deposited ligand structures where > > either there is clearly no density for the ligand, or the > ligand is in > > the wrong place, or even the wrong ligand has been built, > can be laid at > > the door of the test set that was used. Plain incompetence and/or > > wishful thinking is a much more likely explanation. So personally I > > think you're worrying over nothing. > > > > Just my 2p's worth. > > > > Cheers & Seasons greetings to Steve & Jon! > > > > -- Ian > > > > > >> -----Original Message----- > >> From: [log in to unmask] > >> [mailto:[log in to unmask]] On Behalf Of Alun R. Coker > >> Sent: 18 December 2008 22:45 > >> To: CCP4 bulletin board > >> Subject: Re: [ccp4bb] Transferring a Free R set. > >> > >> Hi All, > >> > >> I suspect using a new free assignment for each data set is a > >> fairly wide > >> spread mistake, which would be hard to pick up by a reviewer. > >> It would > >> be interesting to survey the pdb for ligand bound structures > >> which have > >> a different free R set from their parent (isomorphous) native > >> structures. I have heard from a colleague involved in testing > >> refinement programs that there is a surprising number of deposited > >> ligand structures where re-refinement shows that the > ligand is built > >> into noise. > >> > >> Would it be fair to say that in the case of a ligand structure, > >> isomorphous with the native structure but where different > R-free sets > >> have been used, we cannot use the R-free to validate > >> refinement of the > >> ligand? My understanding is that it would be contaminated by > >> the phases > >> brought over with the model from the native data set, and is > >> no longer > >> independent, so any ligand density could just arise from over > >> fitting. > >> How could we test/retrieve such a situation? Will omit maps > >> made after > >> a round of REFMAC refinement without the ligand still have a > >> memory of > >> our over fitted model which would persist through to new > maps? If so, > >> would this be the case after a slow cool in CNS? > >> > >> Does the community still think it is valid to use a slow cool > >> to "reset" > >> the free R? > >> > >> Alun. > >> > >> -- > >> Alun R. Coker > >> University College London > >> Division of Medicine, Royal Free Campus > >> Centre for Amyloidosis and Acute Phase Proteins > >> Rowland Hill Street > >> London > >> NW32PF > >> > >> Tel: +44(0)20 7433 2764 > >> Fax: +44(0)20 7433 2776 > >> > >> > >> > > > > > > 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 > > > > -- > Alun R. Coker > University College London > Division of Medicine, Royal Free Campus > Centre for Amyloidosis and Acute Phase Proteins > Rowland Hill Street > London > NW32PF > > Tel: +44(0)20 7433 2764 > Fax: +44(0)20 7433 2776 > > 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