Re: [ccp4bb] Rfree flag
Dear All, Thank you Folmer and Fred for your suggestions. Mine is an isomorphous crystal. I had refined without using the same set of Rfree reflections, finally getting larger difference between the R and Rfree. I hope considering the same Rfree set would solve this problem. Thank you With regards Kavya Although it is not mandatory, I think it would be a very good idea, especially if you have the exact same spacegroup and the native and ligand-bound forms of your protein are essentially the same. If you do not use the same reflections, you are actually not reporting an independent (free) R-factor. Best regards, Folmer Hello, I think this depends on the type of problem you are facing: if the 2 crystals are not isomorphous then you cannot have the same R-free sets; if the 2 crystals are isomorphous then either you do not worry about keeping the same R-free set (but then the starting structure must be perturbed enough to get rid of R-free bias during refinement), or you keep the same indices for the reflections in the 2 R-free sets (same reflections in your message) in which case there is no R-free bias at the beginning of refinement. By R-free bias I mean this: in your new (liganded) crystal form, there are reflections that have seen the Fo's during refinement of the native structure but that are in the R-free set in the ligand structure. This leads to bias. HTH, Fred. Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Rfree flag
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Kavya, As far as I understand the PDBRedo project attempts to make the reflections unbiased from the structure by a random shift of coordinates (e.g. 'NOISE' keyword in pdbset, although I am not aware of an investigation about whether this actually does make remove bias. It is safest to keep the same Rfree-set. Regards, Tim On 02/28/2013 06:54 AM, Kavyashree Manjunath wrote: Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRLyQ2UxlJ7aRr7hoRAsHBAJ43K7f2lcSZwm6fD1pH8+grvrOqRACg1bdi PUUKJaUv8C4JgmcPNM6H9+U= =J47i -END PGP SIGNATURE-
Re: [ccp4bb] Rfree flag
Hi Tim, Our approach is a bit different. We first try to establish whether the R-free set is biased, by checking whether R-free is surprisingly low compared to R given the data parameter ratio. If this is the case (or if we chose a new R-free set for some reason, e.g. because it was too small) PDB_REDO resets the B-factors to 0.5*(Wilson B-factor) and then does more cycles of refinement to ensure it converges. This should get rid of the bias. We compared this to the 'perturb coordinates' approach and in most cases there wasn't any difference. In some cases the perturbed coordinates were outside the radius of convergence of Refmac (the version 5.2 or perhaps 5.4) particularly in cases with NCS. So coordinate perturbation was just not worth it. This was before NCS restraints were properly implemented in PDB_REDO (hurray for local NCS/LSSR!), so this issue must be much smaller now. Of course the rebuilding round of PDB_REDO followed by more refinemnt, will cause enough model perturbation if the above results are not convincing enough ;) We also do full (well, k-fold) cross-validation for small data sets in which the different test sets are all-but-one completely biased. Here, too, the B-factor resetting works well enough. That said we might add a few extra cycles of refinement here to be on the safe side. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Thursday, February 28, 2013 10:33 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rfree flag -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Kavya, As far as I understand the PDBRedo project attempts to make the reflections unbiased from the structure by a random shift of coordinates (e.g. 'NOISE' keyword in pdbset, although I am not aware of an investigation about whether this actually does make remove bias. It is safest to keep the same Rfree-set. Regards, Tim On 02/28/2013 06:54 AM, Kavyashree Manjunath wrote: Dear users,y Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRLyQ2UxlJ7aRr7hoRAsHBAJ43K7f2lcSZwm6fD1pH8+grvrOqRACg1bdi PUUKJaUv8C4JgmcPNM6H9+U= =J47i -END PGP SIGNATURE-
[ccp4bb] Rfree flag
Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Rfree flag
Dear Kavya, It is not so. It is not mandatory. best, Sekar Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. Could you kindly confirm the safe receipt of this mail please All best wishes and regards, Yours sincerely, Dr. K. Sekar, Ph.D. Associate Professor Bioinformatics Indian Institute of Science Bangalore 560 012 INDIA E-mail: se...@physics.iisc.ernet.in Tel: 91-(0)80-22933059, 23601409, 22932469 Fax:91-(0)80-23600683, 23600551 Homepage: http://www.physics.iisc.ernet.in/~dichome/sekhome/index.html -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Rfree flag
Hello, I think this depends on the type of problem you are facing: if the 2 crystals are not isomorphous then you cannot have the same R-free sets; if the 2 crystals are isomorphous then either you do not worry about keeping the same R-free set (but then the starting structure must be perturbed enough to get rid of R-free bias during refinement), or you keep the same indices for the reflections in the 2 R-free sets (same reflections in your message) in which case there is no R-free bias at the beginning of refinement. By R-free bias I mean this: in your new (liganded) crystal form, there are reflections that have seen the Fo's during refinement of the native structure but that are in the R-free set in the ligand structure. This leads to bias. HTH, Fred. On 28/02/13 06:54, Kavyashree Manjunath wrote: Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] Rfree flag
Although it is not mandatory, I think it would be a very good idea, especially if you have the exact same spacegroup and the native and ligand-bound forms of your protein are essentially the same. If you do not use the same reflections, you are actually not reporting an independent (free) R-factor. Best regards, Folmer 2013/2/28 Prof. K. Sekar se...@physics.iisc.ernet.in Dear Kavya, It is not so. It is not mandatory. best, Sekar Dear users, Is it mandatory to use the same reflections for Rfree calculations of a ligand bound data as that of its native? Thank you With Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. Could you kindly confirm the safe receipt of this mail please All best wishes and regards, Yours sincerely, Dr. K. Sekar, Ph.D. Associate Professor Bioinformatics Indian Institute of Science Bangalore 560 012 INDIA E-mail: se...@physics.iisc.ernet.in Tel: 91-(0)80-22933059, 23601409, 22932469 Fax:91-(0)80-23600683, 23600551 Homepage: http://www.physics.iisc.ernet.in/~dichome/sekhome/index.html -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- Folmer Fredslund
