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CCP4BB  July 2020

CCP4BB July 2020

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Subject:

Re: Problems with refinement of nucleic acid structure

From:

Tim Gruene <[log in to unmask]>

Reply-To:

Tim Gruene <[log in to unmask]>

Date:

Thu, 30 Jul 2020 21:26:50 +0200

Content-Type:

multipart/signed

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Parts/Attachments

text/plain (169 lines) , application/pgp-signature (169 lines)

Dear Rafal,

with such a small molecule, and if you really have 1.0A resolution, you
should be able to solve the structure by direct methods with shelxt. By
default, shelxt searches all space groups in the Laue group. The output
will not be very practical to assign residue, etc. but at least it
might give you a clue where the problem is.

Best regards,
Tim

On Thu, 30 Jul 2020 14:05:54 +0200
Rafal Dolot <[log in to unmask]> wrote:

> 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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-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A

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