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
