Dear Stephen,

                        I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The "dimers" were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers.

Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%.

More details are in Cabezon et al., NSMB 10, 744-750, 2003

Best wishes,

Andrew



On 11 Mar 2014, at 14:04, Stephen Cusack <[log in to unmask]> wrote:

> Dear All,
>    I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated
> in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9.
> Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but  there is a lot
> of extra positive density in the solvent regions (some of it looking like weak density for helices and strands)  and unexpected positive peaks within the model region.
> Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly.
> The two incompatible solutions are related by a 2-fold axis parallel to a.
> This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality
> of pseudo-merohedral twinning (-h, l, k) do not reveal
> any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a
> non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference
> since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions.
> How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ?
> thanks for your suggestions
> Stephen
> 
> -- 
> 
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> Dr. Stephen Cusack,	
> Head of Grenoble Outstation of EMBL
> Group leader in structural biology of protein-RNA complexes and viral proteins
> Joint appointment in EMBL Genome Biology Programme
> Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI)
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