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
