Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be
crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from
data reduced in higher symmetry, because you would be enforcing that higher symmetry.
But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90,
merging statistics would have prevented reducing the data in p222 in the first place.
Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy,
so that on the average it does obey the higher symmetry? Like our "heme on a special position" in the bacterioferritin
paper?
And structure factors add because the two orientations are present in the same domain, whereas with twinning the two
orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on
the "film"?
[log in to unmask] wrote:
> If it is crystal packing disorder (F's added instead of I's), the switches between the alternative conformations need to be very frequent, to be within coherent range, so I would asume that the alternative conformations will be present in equal proportions. Still the alternatives need to be modeled somehow and if this can be conveniently done in a lower symmetry spacegroup this would not artificially lower the free R-factors. As Phoebe mentioned, ignoring the higher symmetry relations and repicking the free Rflags at lower symmetry would lead free reflections to be linked to the working set, leading to too low Rfree values. However, with perfect packing disorder, no extra information would be gained by reprocessing in lower symmetry (in contrast to cases with pseudo symmetry).
>
> My 2 cents,
> Herman
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Phoebe A. Rice
> Sent: Tuesday, March 19, 2013 4:49 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree
>
> oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that "perfect twinning" comment in my last message.
> ________________________________________
> From: CCP4 bulletin board [[log in to unmask]] on behalf of Phoebe A. Rice [[log in to unmask]]
> Sent: Tuesday, March 19, 2013 10:34 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree
>
> Hi Zbyszek,
> If the issue is perfect twinning, I agree - good point!
> But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group.
> Phoebe
>
> ++++++++++++++++++++++++++++++++++++++++++
>
> Phoebe A. Rice
> Dept. of Biochemistry& Molecular Biology The University of Chicago
> 773 834 1723; [log in to unmask]
> http://bmb.bsd.uchicago.edu/Faculty_and_Research/
> http://www.rsc.org/shop/books/2008/9780854042722.asp
>
> ________________________________________
> From: CCP4 bulletin board [[log in to unmask]] on behalf of Zbyszek Otwinowski [[log in to unmask]]
> Sent: Tuesday, March 19, 2013 9:37 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree
>
> It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212).
>> From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning.
>
> Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder.
>
> !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry!
>
> Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry.
>
> Zbyszek Otwinowski
>
>> Dear all,
>> We have solved the problem. Data processing in P1 looks better (six
>> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three
>> molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first
>> round of refinement (without put waters, ligands, etc.).
>>
>> Indeed, there were one more molecule in ASU, but the over-merged data
>> in an orthorhombic lattice hid the correct solution.
>>
>> Thank you very much for all your suggestions, they were very important
>> to solve this problem.
>>
>> Cheers,
>>
>> Andrey
>>
>> 2013/3/15 Andrey Nascimento<[log in to unmask]>
>>
>>> *Dear all,*
>>>
>>> *I have collected a good quality dataset of a protein with 64% of
>>> solvent in P 2 21 21 space group at 1.7A resolution with good
>>> statistical parameters (values for last shell: Rmerge=0.202;
>>> I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better
>>> than last shell). The structure solution with molecular replacement
>>> goes well, the map quality at the protein chain is very good, but in
>>> the final of refinement, after addition of a lot of waters and other
>>> solvent molecules, TLS refinement, etc. ...
>>> the Rfree is a quite high yet, considering this resolution
>>> (1.77A).(Rfree=
>>> 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a
>>> lower symmetry space group (P21), but I got the same problem, and I
>>> tried all possible space groups for P222, but with other screw axis I
>>> can not even solve the structure.*
>>>
>>> *A strange thing in the structure are the large solvent channels with
>>> a lot of electron density positive peaks!? I usually did not see too
>>> many peaks in the solvent channel like this. This peaks are the only
>>> reason for these high R's in refinement that I can find. But, why are
>>> there too many peaks in the solvent channel???*
>>>
>>> *I put a .pdf file (ccp4bb_maps.pdf) with some more information and
>>> map figures in this link:
>>> https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf*
>>>
>>> *
>>> *
>>>
>>> *Do someone have an explanation or solution for this?*
>>>
>>> * *
>>>
>>> *Cheers,*
>>>
>>> *Andrey*
>>>
>>
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385
> Fax. 214-645-6353
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