Some answers: Re FreeR increasing? 1) Have you been careful to keep the same FreeR set for the REFMAC and CNS refinements? I suspect not and that explains why your first FreeR is unrealistically low, and then increases to a more realistic value.
If you use the GUI and select "Create mtz file" from the Reflection Utilities you can ask to import a CNS and preserve the FreeR flags. An easier way is to use this new program from Kevin Cowtan: He says in an earlier email Here's an updated version of cns2mtz. The new version automatically merges anomalous data, generates a full set of CCP4-style free sets from the CNS test column, completes the data (unless you tell it not to), and can take the cell and spacegroup information from a pdb file as well as from the command line. It converts every file I have automatically, but I haven't tested the output extensively. More testing would be good. Mail me with any problems. x86 Linux binary available from here: http://www.ysbl.york.ac.uk/~cowtan/cns2mtz/cns2mtz.html Q2) Then re TLS - you must select and define your own TLS groups Ethan Merritt has a informative paper about how to do this, and a server which make suggestions: Email from Ethan: On Thursday 21 December 2006 10:39, U Sam wrote: > > I would like to know > > (a) how TLS parameters are calculated. > Willis, B. T. M. & Pryor, A. W. (1975). Thermal Vibrations in Crystallography. Cambridge University Press. Winn, M. D., Isupov, M. N. & Murshudov, G. N. (2001). Use of TLS parameters to model anisotropic displacements in macromolecular refinement Acta Cryst. D57, 122�C133. J Painter & E A Merritt (2006) Acta Cryst. D62, 439-450 Optimal description of a protein structure in terms of multiple groups undergoing TLS motion <http://skuld.bmsc.washington.edu/~tlsmd/Acta_D62_439.pdf> Q3) I distrust the stt - R and Rfree should differ by < 3% - this depends on the data you are using as well as the quality of your model.. If you have very high resolution that is likely. If you have NCS you may achieve it but it is somewhat unrealistic If you have 2.6A data you would expect a greater difference.. and so on.. Eleanor zhongzhou chen wrote: > Dear Sir or madam, > > I have some questions about refmac5. > > I am new to Refmac. I optimized the structure with CNS (Rwork: 23%, Rfree: > 25%). > > Cell : 71.480 71.480 151.813 90.000 90.000 90.000 > Space group p43212 > resolution 100 1.97 > > protein contain 350 amino acids. > Nmol/asym: 1 > > number of amino acids built: 330 > number of waters:330 > three glycerols > six sulfate ions. > > > > My friends told me that refmac5 will get lower Rfree and Rfactor. > > I refined the pdb structure from CNS using restrained refinement. > > Ncyc Rfact Rfree FOM LLG rmsBOND rmsANGLE rmsCHIRAL $$ > $$ > > using TLS: > 0 0.215 0.221 0.763 165320.6 0.006 1.250 0.092 > 1 0.189 0.230 0.751 162623.7 0.022 1.680 0.116 > 2 0.187 0.233 0.751 162500.2 0.022 1.724 0.119 > 3 0.185 0.239 0.747 162269.0 0.022 1.790 0.123 > 4 0.185 0.241 0.748 162314.8 0.022 1.809 0.124 > 5 0.185 0.244 0.744 162248.1 0.023 1.831 0.126 > 6 0.185 0.245 0.744 162353.0 0.023 1.839 0.126 > 7 0.185 0.247 0.743 162226.2 0.023 1.858 0.128 > 8 0.186 0.248 0.741 162430.4 0.023 1.852 0.128 > 9 0.185 0.248 0.741 162300.2 0.023 1.857 0.128 > 10 0.185 0.249 0.740 162317.2 0.023 1.861 0.129 > > > without TLS: > Ncyc Rfact Rfree FOM LLG rmsBOND rmsANGLE rmsCHIRAL $$ > $$ > 0 0.217 0.223 0.764 166172.2 0.006 1.250 0.092 > 1 0.192 0.234 0.752 163514.5 0.022 1.676 0.116 > 2 0.190 0.237 0.752 163400.6 0.022 1.715 0.119 > 3 0.188 0.242 0.747 163218.2 0.022 1.783 0.124 > 4 0.188 0.244 0.747 163276.6 0.022 1.802 0.125 > 5 0.188 0.248 0.744 163167.9 0.023 1.828 0.127 > 6 0.188 0.248 0.744 163271.0 0.023 1.832 0.127 > 7 0.188 0.251 0.743 163144.5 0.023 1.849 0.128 > 8 0.189 0.251 0.742 163331.2 0.023 1.845 0.129 > 9 0.188 0.252 0.741 163209.3 0.023 1.850 0.129 > 10 0.188 0.253 0.741 163249.6 0.023 1.854 0.129 > > > May I ask some questions? > > 1. Can I use the structure in zero cycle to deposit in pdb bank? > Because its rfree is lowest and it just come from CNS result without any > change of B > > factor? > > 2. Why Rfree increase during refinement? > > 3. Usually, delta(Rwork,Rfree) is lower than 3.0 percent for a good > structure. > However, there is 7% if using the final refinement structure. > How can I do to reduce Rfree? > > 4. I use the TLS to refine during restrained refinement. Why the result > structure show > > that there is no tls groups? > > The TLS format > RANGE 'A 11.' 'A 60.' ALL > ORIGIN -5.756 -6.929 21.439 > T -0.0229 -0.0472 -0.0103 -0.0487 -0.0019 0.0414 > L 0.8419 0.3881 3.8499 -0.2446 -1.1643 0.8341 > S -0.0615 -0.0707 -0.0949 -0.1532 0.1007 0.1020 0.2434 -0.2249 > > > > The pdb output file is shown as below. > > REMARK 3 TLS DETAILS > REMARK 3 NUMBER OF TLS GROUPS : NULL > > > > thanks > > > Best regards, > > zhong chen > [EMAIL PROTECTED] > 2007-07-12 > > > > This communication is for the use of the intended recipient only. It may > contain information that is privileged and confidential. If you are not the > intended recipient of this communication, any disclosure, copying, further > distribution or use thereof is prohibited. If you have received this > communication in error, please advise me by return e-mail or by telephone and > delete/destroy it. > > >
