[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] intensity statistics and twinning

[Hillen, Hauke]

Agreed, and this is ongoing work. Yet it is not trivial to get better 
resolution than this for small (in the world of EM) samples either.

Von: Mark van Raaij [mjvanra...@cnb.csic.es]
Gesendet: Donnerstag, 27. Oktober 2016 22:08
An: Hillen, Hauke
Betreff: Re: [ccp4bb] AW: [ccp4bb] intensity statistics and twinning

Another option might be to go full cryo-EM

Mark J van Raaij
CNB-CSIC
www.cnb.csic.es/~mjvanraaijOn 27 Oct 2016 21:37, "Hillen, Hauke" 
<hauke.hil...@mpibpc.mpg.de> wrote:
>
> Dear Mark,
>
> Thanks for your reply. Unfortunately, I have already spent a lot of time 
> trying to do exactly this. I do think some biologically relevant questions 
> can be answered by architecture at this resolution, so I would like to try to 
> get the most out of these datasets I can.
>
> Best wishes,
> Hauke
>
>
> 
> Von: Mark van Raaij [mjvanra...@cnb.csic.es]
> Gesendet: Donnerstag, 27. Oktober 2016 21:29
> An: Hillen, Hauke
> Betreff: Re: [ccp4bb] intensity statistics and twinning
>
> I'd say intensity statistics at this resolution at not reliable and your 
> crystal are most likely not twinned.
> Unless you think you can answer any interesting biological question at this 
> resolution, I'd forget about these datasets and spend all my efforts at 
> getting better diffracting ones. Even if this means going back to cloning or 
> working on a different species...although lysine methylation, limited 
> proteolysis etc. might also be tried if you haven't done so already.
> A slightly different crystal contact might make the difference and yield much 
> better diffraction.
>
> Mark J van Raaij
> CNB-CSIC
> www.cnb.csic.es/~mjvanraaij
>
> On 27 Oct 2016 21:11, "Hillen, Hauke" <hauke.hil...@mpibpc.mpg.de> wrote:
> Dear ccp4 community,
>
> I am currently working on some low resolution datasets (around 4.5A). The 
> space group seems to be P21, as suggested by XDS and pointless. I have 
> collected many datasets of these crystals, both native as well as 
> SeMet-labeled. Using MR-SAD I have been able to obtain a clearly 
> interpretable electron density map for all features I expect and heavy atom 
> sites that make sense for both the model used in MR and the yet unmodeled 
> components. So far, so good.
>
> While routinely analyzing my datasets with Phenix Xtriage, I have noticed 
> that the intensity statistics for all of these datasets look unusual. In 
> fact, Xtriage complains about this with the message: „The intensity 
> statistics look unusual, but twinning is not indicated or possible in the 
> given space group“ when processed in P21.
> The occurence of this message depends somewhat on the typ of input file I use 
> for the same dataset as well as the input parameters (high resolution 
> cut-off). If I use XDSCONV to convert the intensities to amplitudes for 
> phenix, this message appears. If I use the output of XSCALE directly as 
> intensities, this message does not appear, yet the actual statistics are 
> somewhat similar. I have attached the log file output for four scenarios at 
> the end of this message (P21 intensities, P21 amplitudes, P1 intensities, P1 
> amplitudes).
> These results got me questioning whether the true space group is really P21, 
> or whether it could be that it is P1 with some twinning issue. Since the 
> Xtriage output regarding the „normality“ of the intensity statistics varies 
> upon the input format, I assume that this case may be somewhat borderline. 
> Since I have very little experience both with low-resolution crystals as well 
> as with twinning, I am a bit unsure how to proceed with this data.
> How can I distinguish between a partially twinned P1 crystal and an untwinned 
> P21 crystal? It is my impression from previous discussions here that 
> distinguishing twinned from untwinned data simply by comparing refinement 
> results with and without twin laws is not always conclusive, as the R-factors 
> are not directly comparable. If the crystal is truly P21, could these issues 
> arise from intensity to amplitude conversion problems? (Xtriage also suggests 
> this as a possibility) If so, can these be overcome? Or could the deviation 
> from ideal intensities simply originate from the low quality (= resolution) 
> of the data and are within the range of tolerance for such a dataset? Could 
> this be some type of pseudosymmetry issue? And finally, what
>
> I would be very grateful for any advice on how to proceed with these data!
>
>
> Kind regards,
> Hauke
>
>
>
> Processed as P21, intensity input:
>
> === Diagnostic tests for twinning and pseudosymmetry 
> ==
>
> Using data 

[ccp4bb] AW: [ccp4bb] intensity statistics and twinning

[Hillen, Hauke]

Dear Mark,

Thanks for your reply. Unfortunately, I have already spent a lot of time trying 
to do exactly this. I do think some biologically relevant questions can be 
answered by architecture at this resolution, so I would like to try to get the 
most out of these datasets I can.

Best wishes,
Hauke



Von: Mark van Raaij [mjvanra...@cnb.csic.es]
Gesendet: Donnerstag, 27. Oktober 2016 21:29
An: Hillen, Hauke
Betreff: Re: [ccp4bb] intensity statistics and twinning

I'd say intensity statistics at this resolution at not reliable and your 
crystal are most likely not twinned.
Unless you think you can answer any interesting biological question at this 
resolution, I'd forget about these datasets and spend all my efforts at getting 
better diffracting ones. Even if this means going back to cloning or working on 
a different species...although lysine methylation, limited proteolysis etc. 
might also be tried if you haven't done so already.
A slightly different crystal contact might make the difference and yield much 
better diffraction.

Mark J van Raaij
CNB-CSIC
www.cnb.csic.es/~mjvanraaij

On 27 Oct 2016 21:11, "Hillen, Hauke"  wrote:
Dear ccp4 community,

I am currently working on some low resolution datasets (around 4.5A). The space 
group seems to be P21, as suggested by XDS and pointless. I have collected many 
datasets of these crystals, both native as well as SeMet-labeled. Using MR-SAD 
I have been able to obtain a clearly interpretable electron density map for all 
features I expect and heavy atom sites that make sense for both the model used 
in MR and the yet unmodeled components. So far, so good.

While routinely analyzing my datasets with Phenix Xtriage, I have noticed that 
the intensity statistics for all of these datasets look unusual. In fact, 
Xtriage complains about this with the message: „The intensity statistics look 
unusual, but twinning is not indicated or possible in the given space group“ 
when processed in P21.
The occurence of this message depends somewhat on the typ of input file I use 
for the same dataset as well as the input parameters (high resolution cut-off). 
If I use XDSCONV to convert the intensities to amplitudes for phenix, this 
message appears. If I use the output of XSCALE directly as intensities, this 
message does not appear, yet the actual statistics are somewhat similar. I have 
attached the log file output for four scenarios at the end of this message (P21 
intensities, P21 amplitudes, P1 intensities, P1 amplitudes).
These results got me questioning whether the true space group is really P21, or 
whether it could be that it is P1 with some twinning issue. Since the Xtriage 
output regarding the „normality“ of the intensity statistics varies upon the 
input format, I assume that this case may be somewhat borderline. Since I have 
very little experience both with low-resolution crystals as well as with 
twinning, I am a bit unsure how to proceed with this data.
How can I distinguish between a partially twinned P1 crystal and an untwinned 
P21 crystal? It is my impression from previous discussions here that 
distinguishing twinned from untwinned data simply by comparing refinement 
results with and without twin laws is not always conclusive, as the R-factors 
are not directly comparable. If the crystal is truly P21, could these issues 
arise from intensity to amplitude conversion problems? (Xtriage also suggests 
this as a possibility) If so, can these be overcome? Or could the deviation 
from ideal intensities simply originate from the low quality (= resolution) of 
the data and are within the range of tolerance for such a dataset? Could this 
be some type of pseudosymmetry issue? And finally, what

I would be very grateful for any advice on how to proceed with these data!


Kind regards,
Hauke



Processed as P21, intensity input:

=== Diagnostic tests for twinning and pseudosymmetry ==

Using data between 10.00 to 3.50 Angstrom.

--Patterson analyses--

Largest Patterson peak with length larger than 15 Angstrom:
Frac. coord.  :0.1640.000   -0.021
Distance to origin:   17.720
Height relative to origin :3.072 %
p_value(height)   :1.000e+00

Explanation
The p-value, the probability that a peak of the specified height or larger
is found in a Patterson function of a macromolecule that does not have any
translational pseudo-symmetry, is equal to 1.000e+00.  p_values smaller than
0.05 might indicate weak translational pseudo symmetry, or the self vector of
a large anomalous scatterer such as Hg, whereas values smaller than 1e-3 are
a very strong indication for the presence of translational pseudo symmetry.


 --Wilson ratio and moments--

Acentric reflections:


  /^2:1.935   (untwinned: 2.000; perfect twin 1.500)
  ^2/:0.805   (untwinned: 0.785;