I guess my experience is out of date - please ignore comments on LLG! Eleanor
On 21 October 2013 15:40, Randy Read <rj...@cam.ac.uk> wrote: > Hi Eleanor, > > Yes, the initial LLG scores in Phaser are highly dependent on the assigned > sequence identity, which is translated into an initial estimate of the > effective RMSD of the model. However, the latest versions of Phaser refine > the RMSD estimate at the end of the job and, assuming that two runs find the > same solution and the refinement manages to converge (which it usually does > these days), the LLG at the end should be pretty reproducible regardless of > the assigned sequence identity. > > Best wishes, > > Randy > > On 21 Oct 2013, at 14:42, Eleanor Dodson <eleanor.dod...@york.ac.uk> wrote: > >> I dont know about LLG scores - they seem extremely variable depending >> on the degree of sequence similarity you assign. >> However when you get an R/Rfree of 40%/47% that is a pretty good sign >> that at least something is correct. >> >> It isnt clear whether that is after you have placed 2 copies of the >> second component? >> Anyway - maybe try again with the refined component 2 and look for >> component 1 again? >> Eleanor >> >> >> >> On 18 October 2013 15:51, <herman.schreu...@sanofi.com> wrote: >>> Dear Jan, >>> >>> There are a few things a would do in this case. The first is to check the >>> processing to make sure the space group is really C2 and, although >>> unlikely, not some other space group. >>> >>> The second thing would be to try to place the first component. From your >>> email it is not clear to me whether or not you were able to place the first >>> component after the second component had been placed. In your case, I would >>> give both components to phaser and ask phaser to first place component 2 >>> and then component 1. >>> >>> It might be that the correct solution gets rejected because of clashes, so >>> I would also try to trim the first component, or to increase the number of >>> allowed clashes in Phaser. Although you expect two copies of your >>> heterodimer, you may have a crystal with a high solvent content and only >>> one dimer in the asymmetric unit. In this case the crystal packing should >>> make sense i.e. continuous crystal contacts in all three dimensions. >>> >>> Best, >>> Herman >>> >>> >>> -----Ursprüngliche Nachricht----- >>> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan >>> Félix >>> Gesendet: Freitag, 18. Oktober 2013 13:17 >>> An: CCP4BB@JISCMAIL.AC.UK >>> Betreff: [ccp4bb] Molecular Replacement using low sequence identity >>> templates >>> >>> Dear all, >>> I have a question regarding Molecular Replacement using low sequence >>> identity templates. >>> >>> I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex >>> (space group C 1 2 1, no twinning detected using xtriage). For the first >>> component homologs are available, but for the other the best found template >>> only has 20 % sequence similarity. >>> Strangely, I cannot place the first component directly, but the second >>> component can be placed (after trimming the template with chainsaw) using >>> phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 >>> NCS copies of the heterodimer are expected based on the unit cell >>> parameters, only 1 copy of the second component gets placed. >>> If I try to place the first component based on the .sol file of the first >>> MR solution, the TFZ score for the second placement is only about 3.5, but >>> if I then try to place this second MR solution (2 >>> components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000. >>> >>> However, none of the MR solutions I obtained seems to refine in PHENIX. >>> Using autobuild does not lower the R/Rfree values which seem to get stuck >>> at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated >>> annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to >>> improve the model.. >>> Also, in every MR solution only half of the asymmetric unit seems to be >>> filled, but phaser fails to place more units..As I am seriously starting to >>> doubt the actual content of the crystals, I tried Nearest Cell to search >>> for similar space group, but without any hits. >>> >>> So here is my question. Is it possible to get TFZ/LLG values this high in >>> C 1 2 1 with a completely incorrect model by chance, or can I assume that >>> this MR solution points out that what I think is in the crystal is actually >>> there? >>> And secondly, as I am a bit stuck here, are there any new strategies I can >>> try to tackle this problem? >>> Off course, experimental phasing is an option, but the crystals grew slowly >>> over e few months and I only had 1 drop with 1 crystal, so reproducing the >>> crystals might be though.. >>> >>> Thanks for any tips and best regards, >>> >>> Jan > > ------ > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: + 44 1223 336500 > Wellcome Trust/MRC Building Fax: + 44 1223 336827 > Hills Road E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk >
