In situations like this I always try dropping to P1. Even if the data
are highly incomplete in P1 you can still refine it. Difference maps are
degraded by poor completeness, but you still might see something. But
either way, the R-factors will tell you something. if dropping to P1
solves your R-factor problems then you know you were in the wrong space
group.
The biggest caveat to dropping symmetry operators (and essentially
replacing them with NCS operators) is that you want to be VERY careful
not to mix your working and free sets. The best way to do this is pick
your free set with the highest-possible symmetry given your lattice, and
then expand that to P1 using the CCP4 program "CAD". Then you change
the space group in "CAD" and it will chunk out the right asymmetric
unit. Oh, and then use PDBSET to expand your model to P1 as well.
All this is done automatically by the program Zanuda, but even Zanuda
cannot un-merge data. You need to provide the P1 structure factors and
then see what it tells you.
Does that help?
-James Holton
MAD Scientist
On 4/18/2017 5:56 PM, gnufreebsd wrote:
Dear Pravin
for a kinase, n lobe is quite flexible , especially beta1 and beta2,
and residues beyond theses two strands.
best regards
tiger
发自 WPS邮箱客戶端 <http://mo.wps.cn/wps-mail/mail.html>
在 Pravinkumar Jagtap <pravinja...@gmail.com>，2017年4月12日
上午2:55写道：
Dear All,
I am stuck with this problem for 2 months and hope you could help.
We have a 2.1 A dataset for a 380 amino acid long protein. The
space group is I4 (single molecule in asymmetric unit, 48% solvent
content) and the dataset is quite perfect (no obvious
pathologies). The protein itself is organised in 2 lobes (N and C
terminal lobes). The sequence identity to nearest homologue
structure is 17%.
We could get the phases by SeMet SAD phasing (3A resolution
dataset, 5 SeMet (excluding N-terminal Met), 3 full occupancy
SeMet in C-terminal lobe and 2 partial occupancy (~0.5 each;
present on surface) SeMet in N-terminal lobe). Automated model
building (at 2.1 A) yielded nice model for the C-terminal lobe
(215 residues) and manually I could build parts (around 80
residues) of N-terminal lobe with high confidence. In addition we
could also build a ligand which is sandwiched between C and N
terminal lobe.
However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed
some patchy density left at the N terminal lobe but as it is
discontinuous, I cannot build anything in it (except lots of water
molecules). In total I am missing around 85 residues. These
residues are predicted to be present in secondary structure (and
not flexible).
As I have around 75-80% model built, I would expect that I would
have all the phases and should get nice density for the remaining
part. But as I dont see it, could the rest part be flexible? But
again, this is not reflected in the R factors (I would then expect
low Rfree).
Could it be that I still lack phases (due to partial occupancy of
SeMeth in N-terminal lobe ) and have to try to get them by heavy
metal soaking, or there is disorder in the N-terminal lobe? I have
also tried solving different datasets for same crystal but this
has not been useful.
Regards,
Pravin.
