Dear Mark,
In this case you will have to apply Baysian statistics: given the prior: same protein, same space group same cell dimensions and molecular replacement fails completely, the likelihood of having some depressing coincidence somewhere is approaches 100%!
What I would do in addition to excellent suggestions you already got, is to try to download the Fobs from the pdb for the structures with the same protein, space group and cell dimensions, and calculate pattersons with those. Sometimes strong peaks appear in pattersons for no obvious reasons.
I would also consider statistical disorder, which will not show up in twinning statistics since in this case F's (including phases) are added instead of I's. Anyways, it will be an interesting puzzle to solve!
Good luck,
Herman
-----Ursprüngliche Nachricht-----
Von: CCP4 bulletin board [mailto:[log in to unmask]] Im Auftrag von Mark Wilson
Gesendet: Donnerstag, 3. September 2015 02:06
An: [log in to unmask]
Betreff: Re: [ccp4bb] Translational NCS with one molecule in ASU
Dear CCP4 Community,
I've had a number of helpful responses (on- and off-list) that I will briefly summarize via response, including information that I probably should have included in the original post. Many have suggested a wrong space group, which I agree seems probable. MR was attempted in PHASER with all possible choices of space group for a primitive orthorhombic lattice, and in all cases failed with no rotation or translation peaks above a Z-score of 5.
I've not yet tried monoclinic lattices and will, but this still wouldn't explain (to me anyway) an apparently impossible combination of translational NCS in P212121 with a cell that can't accommodate a second molecule unless twinning was also present, which may be the case (as Eleanor suggested). Others have asked about evidence of missed weak reflections indicating a larger true cell, which I looked for but didn't see in these images. The crystal that was used was mounted at room temperature, so there is no opportunity for cryo artifacts to have done something strange to the cell.
Other suggestions included the presence of strong internal symmetry in the molecule, which is present, but as a pseudo-threefold, which seems incompatible with my NCS centering operation. One respondent suggested that we've crystallized the wrong molecule, which is something I also worried about a bit. Although possible, the space group and cell for our crystal are both as previously reported for this protein by another group, so it would be a depressing coincidence if we crystallized the wrong protein in the same cell. I'll be happy to update if/when we figure this out should it be of interest to the board. Thank you all for your thoughtful responses, which arrived in impressive number in the time it took me to drive home.
Best regards,
Mark
Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
[log in to unmask]
On 9/2/15 5:30 PM, "CCP4 bulletin board on behalf of Eleanor Dodson"
<[log in to unmask] on behalf of [log in to unmask]> wrote:
>Well - a translation of 0 0.5 0 would generate absences along b so
>that the SG could be P212121 or P 21 2 21Ð
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>I would suspect twinning and a monoclinic SG .
>Or as we found sadly - half the protein had disappeared in the
>crystallisation trials..
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>But such a translation must mean you almost have a halved unit cell?
>Another way of saying there isn't enough room for your molecule..
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>On 2 September 2015 at 22:38, Shane Caldwell
><[log in to unmask]> wrote:
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>Are you certain it's actually P212121? One possibility is you're at
>lower symmetry and the Patterson peak corresponds to the NCS between
>particles that are almost-but-not-quite crystallographically
>equivalent. In that case, MR probably wouldn't work. Does searching in
>P1 find anything?
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>Shane Caldwell
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>McGill University
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>On Wed, Sep 2, 2015 at 5:19 PM, Mark Wilson <[log in to unmask]> wrote:
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>Dear CCP4 community,
>I've encountered a curious problem with some recently collected data.
>I have a 1.8 Å resolution dataset that scales well in P212121 (log file
>available upon request). The unit cell parameters are similar to those
>reported for crystals of the same protein in a previous publication,
>although my crystallization condition is different. Nevertheless, my
>data produce a strong (47% of origin) peak in the Patterson map at 0.0,
>0.5, 0.0, indicative of translational NCS. However, the unit cell
>parameters can accommodate only one molecule in the ASU without
>single-digit solvent content. Moreover, molecular replacement with a
>model that should be nearly identical fails. Standard intensity-based
>tests show no evidence of twinning or other data pathology. Any thoughts would be appreciated.
>Best regards,
>Mark
>
>Mark A. Wilson
>Associate Professor
>Department of Biochemistry/Redox Biology Center University of Nebraska
>N118 Beadle Center
>1901 Vine Street
>Lincoln, NE 68588
>(402) 472-3626 <tel:%28402%29%20472-3626> [log in to unmask]
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