STARANISO anisotropy & Bayesian estimation server.

The STARANISO Server

Anisotropy of the Diffraction Limit
and
Bayesian Estimation of Structure Amplitudes

Advice on inclusion of test-set (Rfree) flags

We are grateful to Andrew Leslie (MRC-LMB) for identifying a potentially undesirable 'feature' of CCP4/REFMAC (and possibly other refinement software), namely the default 'fill-in' of unmeasured ('missing') data outside the diffraction cut-off surface with DFcalc. In general, any treatment of STARANISO's output data that adds missing reflections out to the diffraction limit may result in a very high proportion of unobserved data compared with data that have had a cut-off applied according to an isotropic criterion. Where missing reflections have been added in this way the user should take care to verify that any applications used to post-process the data can handle it correctly.

In order to deal with this problem, an extra column (label = 'SA_flag', type = 'I') has been added to the STARANISO output MTZ file. SA_flag has one of 4 possible values, as follows:

SA_flag value	Point color	Meaning*
0 or MNF (missing)	Grey	*Unobservable* (not measured and not expected to be significant).
1	Dark blue	*Observable* (not measured but expected to be significant).
2	Red or pink	*Unobserved* (measured but not significant).
3	Orange through white	*Observed* (measured and significant).

*See the GLOSSARY for more explanation of the terminology used here.

Note that currently only values 1 and 3 are output and a new checkbox has been added to the data entry page to use this flag. If left checked the uniqueify script is used to add test-set (R_free) flags to the output merged MTZ file from STARANISO out to the isotropic diffraction limit. Then the file is copied, omitting 'unobservable' reflections outside the anisotropic diffraction cut-off surface where SA_flag has a 'missing number' value. The BUSTER structure refinement program (Global Phasing) is 'SA_flag aware' so that manipulation of the SA_flag column is unnecessary (but harmless) provided that BUSTER is the only refinement program that will be used.

Provided the box for adding test-set (R_free) flags is left checked these steps are now performed by the server as described above (unless test-set flags were already present in the merged input data) and therefore additional user action is no longer required.
If the box for adding test-set (R_free) flags is unchecked, the behaviour of the software is as for the previous version and therefore addition of test-set flags becomes the responsibility of the user (again unless test-set flags were already present in the merged input data). This will be the case where the user wishes to add test-set flags from a reference dataset. Unless only the BUSTER refinement program will be used, this must then be followed by removal of reflections with missing SA_flag value, and optionally, for compatibility with some downstream pipelines. removal of the SA_flag column. For this purpose the following script can be used:
```
sftools
> read input.mtz
> select col SA_flag present
> delete col SA_flag    #ONLY if required by downstream pipeline.
> write output.mtz
> end
```
The result of this is that REFMAC (or other refinement software) will only fill in the 'observable' missing reflections (blue points) with DF_calc that lie inside the anisotropic diffraction cut-off surface, which is correct, and not to the 'unobservable' missing reflections (grey points) and 'unobserved' (red or pink) that lie outside the cut-off surface.

The STARANISO Server

Anisotropy of the Diffraction Limitand Bayesian Estimation of Structure Amplitudes

Advice on inclusion of test-set (Rfree) flags

Anisotropy of the Diffraction Limit
and
Bayesian Estimation of Structure Amplitudes