Dear Rafal,
Another few comments from my side:
1) I guess you tried to fit alternative positions for the bases but this did
not work?
2) I also guess you refined already anisotropic B-factors?
3) from the figure you send, the absolute value of the difference density looks
very low (maybe 10% of the average 2mFo-dFc density). This means that whereas
some density for the bases shows up, you may not see very much for the rest of
the molecule and the phosphates may not move.
If you have not yet done it, I would try to fit alternate positions and
otherwise leave it as it is.
Best,
Herman
-----Ursprüngliche Nachricht-----
Von: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Im Auftrag von Rafal Dolot
Gesendet: Donnerstag, 30. Juli 2020 14:06
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Problems with refinement of nucleic acid
structure
EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk
Dear all,
Thank you for the response. I will try to explain it more precisely.
The molecule of interest is a duplex with 9 nt length, but is paired on the
length of eight bases, with overhangs at ends. Molecules form a parallel
strings across the crystal lattice, parallel to C-axis, because of these
stacked overhangs. The structure was solved by MR using Molrep.
Trials using Phaser were failed. The initial model was obtained by ZN-SAD.
Refinement was dome for space group P43212, with cell parameters
31.96 31.96 95.07 90 90 90, with one duplex molecule per AU.
Schreuder, Herman /DE wrote:
> At this resolution, one sees many amino-acid side chains with
> alternative conformation, so it might be a good idea to test if this
> is also true for nucleotides.
Dear Herman,
I'm working on some protein ultra-high resolution structures (around 1.0 A or
higher), and alternative conformations are nicely visible on electron density
maps. In this case, there is visible almost all molecule, when you switch
contouring to 2 sigma or lower on Fo-Fc maps, so I think, in this case it's not
the same situation.
Matthew Snee wrote:
> Maybe test the spacegroup with Zanuda, and reprocess with the most
> likely lower symmetry group.
> I guess the stats should improve if you identify a pseudo symmetry
> operator that is currently being treated as a true symmetry operator?
Eleanor Dodson wrote:
> Sometimes ghost like this mean there is a spacegroup error - absences
> can be the result of the non-crystallographic translation and not be
> truly indicitive of the spacegroup. What is the possible spacegroup
> and what is the NC translation vector?
Dear Matthew and Eleanor,
I run Zanuda on my datasets, and the output (which is below) suggested, that
spacegroup is right chosen.
Step 1.
R-factors for the starting model.
Transformation into a supergroup.
---------------------------------------------------------------------
| Subgroup | Spacegroup | R.m.s.d. | Refinement in tested group |
| | | from the |--------------------------------|
| Ref | | starting | Rigid | Restrained |
| | | model, A |----------|---------------------|
| | | | R | R | R-free |
|----------|------------|----------|----------|----------|----------|
| >> 10 | P 43 21 2 | 0.0002 | -- | 0.5107 | 0.4871 |
| 10 | P 43 21 2 | 0.0002 | -- | -- | -- |
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Step 2.
Refinements in subgroups.
There are 8 subgroups to test.
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
| >> 10 | P 43 21 2 | 0.0002 | -- | 0.5107 | 0.4871 |
---------------------------------------------------------------------
| 1 | P 1 | 0.0883 | 0.5252 | 0.4985 | 0.4883 |
| 2 | C 1 2 1 | 0.0828 | 0.5447 | 0.5006 | 0.4877 |
| 3 | P 1 21 1 | 0.0824 | 0.5367 | 0.5018 | 0.4921 |
| 4 | P 1 21 1 | 0.0789 | 0.5292 | 0.4971 | 0.4846 |
| 6 | P 21 21 21 | 0.0956 | 0.5380 | 0.5064 | 0.4929 |
| 7 | P 43 | 0.0935 | 0.5183 | 0.4952 | 0.4835 |
| 9 | C 2 2 21 | 0.0908 | 0.5435 | 0.5042 | 0.4910 |
| 10 | P 43 21 2 | 0.0855 | 0.5427 | 0.5097 | 0.4913 |
---------------------------------------------------------------------
| << 7 | P 43 | 0.0935 | 0.5183 | 0.4952 | 0.4835 |
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Step 3.
Refinement of the best model.
Candidate symmetry elements are added one by one.
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
| >> 7 | P 43 | 0.0935 | 0.5183 | 0.4952 | 0.4835 |
---------------------------------------------------------------------
| 1 | P 1 | 0.0927 | 0.5293 | 0.4991 | 0.4950 |
| 8 | C 1 2 1 | 0.0848 | -- | 0.5017 | 0.4906 |
| 9 | C 2 2 21 | 0.0871 | -- | 0.5059 | 0.4928 |
| 10 | P 43 21 2 | 0.0919 | -- | 0.5180 | 0.5109 |
---------------------------------------------------------------------
| << 10 | P 43 21 2 | 0.0919 | -- | 0.5180 | 0.5109 |
---------------------------------------------------------------------
R-factor in the original subgroup is (almost) the best.
The original spacegroup assignment seems to be correct.
According the non-crystallography translation vector, there is an output from
xtriage:
-----------------------------------------------------
| XYZ | height | p-value(height) |
-----------------------------------------------------
| 0.000, 0.000, 0.334 | 53.049 | 4.456e-05 |
| 0.000, 0.000, 0.167 | 28.966 | 1.681e-03 |
| 0.000, 0.000, 0.500 | 27.692 | 2.102e-03 |
-----------------------------------------------------
Jon Cooper wrote:
> Hello, have you tried anisotropic B-factor refinement? It is usually
> very good at cleaning-up the difference map. At that sort of
> resolution, you may still have some way to go in the refinement since
> the R and R-free usually go to about half of what you have, at least
> with proteins.
Dear Jon. Yes, I used anisotropic refinement as usually for this sort of
resolution (1.4 A or better). But it looks too high for this small object and
very high resolution, in my opinion.
Best regards,
Rafal
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