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The tests that output twinning fractions are *not* diagnostic for twinning; they merely estimate what the twin fraction would be if the data were in fact twinned, which can only be decided on the basis of abnormal intensity statistics.  (Any version of Xtriage since July should state this more clearly, since we've seen so many users make this mistake in the past.)  I'm not sure what you mean by the L-test plot being "sigmoidal" - usually this is a diagnostic feature of the NZ plot.  If you could post images of these plots (Xtriage will let you save them, probably ccp4i loggraph will too), that might help.

Given the fact that it scales in P622 despite the ASU being too small, it may be the case that it really is twinned (I'm not exactly sure how to interpret the Refmac results), but you need to absolutely rule out other possibilities before resorting to twinned refinement.  It is certainly possible to solve such a structure but very tricky to refine without fooling yourself.  At 4Å resolution you will already have a difficult time coping with model bias in the 2Fo-Fc map, and twin refinement will make this even worse (whether or not you actually have twinning).

-Nat




On Wed, Sep 17, 2014 at 11:23 PM, Sudipta Bhattacharyya <[log in to unmask]> wrote:
Dear Community,

Recently, we could solve a structure of DNA/protein complex through MR phasing. The data was initially indexed and scaled in P622 space group, however owing to the incompatibility of a single DNA/protein complex to fit in the ASU, it could not be solved in that space group (according to solvent content analysis). Since it was an indication of possible twining event, we tried MR in P321, P312 and P6 space groups respectively and finally got a very good solution in P65. According to phenix xtriage analysis, the data may be a near perfect merohedrally twinned one (twining fraction 0.425, Britton analysis;  0.468 H test; 0.478 ML method; with a possible twin operator h,-h-k,-l), however, the L test rather suggest no such twining event (but the graph of acentric observed, appeared slightly, sigmoidal compared to the straight acentric theoretical in the L test). The same phenomenon happened when we checked the data for possible twining in Truncate (twin fraction: L test: No; H test, 0.42, Murray Rust, 0.35; Britton ML, 0.47; possible twin operator: h+k,-k,-l).  On the other hand, while refining the data in Refmac5 with intensity based twin option ON, Refmac5 suggested a perfect merohedral twining with the fraction of 0.49/0.50.

In the context of these confusing situation, now my questions are -

1. Is the data twinned or not?
2. With such a high twining fraction, is it solvable?
3. What refinement programs will be the best choices for refinement of such a twinned data?
4. In one of the tutorials of Refmac5 it has been suggested, in such twin refinement cases, to choose Rfree set in higher space group (in our case P6522) then expand it to lower space group (in our case P65), could anybody please let me know how to do that in CCP4 or else?
5. It is a data of 4A resolution could anyone tell me what final R/Rfree one could expect from a 4A data (although it may sound a dumb question...)

Any help will be highly appreciated.

With my best regards,
Sudipta.

Sudipta Bhattacharyya,
Postdoctoral Research Fellow,
Colorado State University, Fort Collins,
Colorado, USA.