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 'fill-in' of unobserved 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 the case of merged data this problem can easily be avoided if, instead of running the uniqueify script (or other method) to add missing reflections with test-set (R_free) flags to the output MTZ file from STARANISO, one runs it first on the input MTZ file and submits the resulting MTZ file to the server. STARANISO will then transfer the input R_free flag column to the output file. This way, the anisotropic cut-off is correctly applied to the missing data and the user can proceed as normal.
In the case of unmerged data being submitted to the server (i.e. the recommended approach), the method for merged data above is not feasible and a different method is needed. To this end, 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
1 Dark blue Observable
2 Red or pink Unobserved
3 Orange through white Observed

*See the GLOSSARY for an explanation of the terminology used here.
Note that currently only values 1 and 3 are output. This means that the uniqueify script (or other method) can again be used, this time on the output MTZ file from STARANISO. It is then necessary to select only reflections with a valid SA_flag value (i.e. the reflections with valid data inside the diffraction cut-off surface). This can easily be achieved using the CCP4 SFTOOLS utility to select the valid reflections from the output file from STARANISO before attempting to use the data in refinement, for example:
sftools
> read input.mtz
> select col SA_flag present
> delete col SA_flag
> 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 diffraction cut-off surface, which is correct, and not to the 'unobservable' missing reflections (grey points) that lie outside the diffraction cut-off surface.
The second method above can also be applied to merged data if test-set flags were not added to the input file to STARANISO.

SA_flag value	Point color	Meaning*
0 or MNF (missing)	Grey	Unobservable
1	Dark blue	Observable
2	Red or pink	Unobserved
3	Orange through white	Observed

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