I dont think you expect much better R factors with twinning or without - the fact is the "crystal" is not perfect . Sometimes it is worth screening other crystals from the batch - smaller ones may not be twinned at all.. Have you looked back atthe images? Gerard suggested there might be lattice translocation.. Eleanor
On Tue, 31 Aug 2021 at 16:46, Peer Mittl <[email protected]> wrote: > Dear Herman, Kay, Eleanor and CCp4C > > At the beginning I was very enthusiastic about the twinning option, but > meanwhile the enthusiasm has vanished. I basically get the same Rfactors > after refining the structure in P32 with 5 chains in Refmac (test > reflections selected in resolution shells with sftools straight from the > beginning). > > Refinement with tight NCS restrains on 4 chains and twinning results in > Rf/Rfree 24.2/30.0, without twinning I get 27.0/32.5. With slightly relaxed > NCS restrains for the side chains and no twinning its 24.3/31.8. A Rfree > difference of 2% is that sufficient to confirm twinning? > > What bothers me even more is the difference ED for the "extra" chain (the > on on the special position in P3221). After refienement with twinning the > difference density is slightly reduced but it is still present. > Furthermore, I can replace the "extra" chain by a 180° rotated copy and the > refinemnt ends up in basically the same Rfactors. I attached two screen > shots from the superposition of the initial structure (all atoms in wheat) > on the rotated structure (blue Ca-trace) with the corresponding ED maps. > > All the best, > Peer > > -----"CCP4 bulletin board" <[email protected]> schrieb: ----- > An: [email protected] > Von: "Schreuder, Herman /DE" > Gesendet von: "CCP4 bulletin board" > Datum: 27.08.2021 16:37 > Betreff: [ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on > 2-fold axis? > > > Dear Lijun and others, > > As Kay rightly pointed out, twinning is fundamentally different from e.g. > disorder or alternative conformations. However, it does not depend on a > chain or side chain rapidly jumping between two states, but whether they > are within coherent length of each other. Since most data are collected > from frozen crystals, no side chain will be rapidly jumping between two > states. They will be frozen either conformation A or conformation B and > since these conformations are randomly distributed throughout the crystal, > they are in general within coherent length of each other and causing > interference of their diffracted X-ray’s, so their complex structure > factors are added. > > However, for twinned crystals, the twin domains (twin pieces as you call > it), behave as independent crystals and their intensities are added and not > their (complex) structure factors. > > I did not realize it when I first looked at the thread but the correct > treatment of these crystals will depend on whether there are twin domains > present in the crystals, or whether the two orientations of the disordered > chain are randomly distributed throughout the crystal. Given that the two > orientations have different twin fractions, my bet is that the twin > supporters are right. > > Best, > Herman > > > > > > Von: Lijun Liu <[email protected]> > Gesendet: Freitag, 27. August 2021 15:57 > An: Schreuder, Herman /DE <[email protected]> > Cc: [email protected] > Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold > axis? > > Dear Herman: > > > > if you say “twinned” chains, then it already means same thing of the two > and the side chain interactions could not be different (as you can see only > one copy in the output coordinates), unless you talking about borders > between twin pieces. > > > > In this situation, the only I could imagine to be if P3221, it would have > to make that chain very very rapidly jump between two states in the same > asu, which can be easily proven wrong. > > > > I do agree if refined under P3221 without restraining the two strictly, > side chain interactions may show differences —— data always not perfect! > > > > Lijun > > Sent from my iPhone > > > > > On Aug 27, 2021, at 8:12 AM, Schreuder, Herman /DE < > [email protected]> wrote: > > > Dear Lijun, > with this argument I agree: the interactions between the two orientations > of the “twinned” chain and the neighboring molecules will be different and > the interacting side chains will almost certainly have different > orientations, which necessitates a twinning of the whole structure. > Best, > Herman > > > > Von: CCP4 bulletin board <[email protected]> Im Auftrag von Lijun Liu > Gesendet: Freitag, 27. August 2021 14:22 > An: [email protected] > Betreff: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold > axis? > > > I believe it is a twin from P32. > > > > > > > > Not like the assignment of double conformations with partial occupancies > to small part of asu, for examples, a side chain of lysin or a small > fragment of a protein, which have both conformations stayed in the same > specific asu at the same time. For this p3221 asu, if one copy of that > chain occupies the part of the asu, the other so-called conformation will > not appear in the same asu at the same time, so the conformation and > occupancy issue was arisen from different cell units, it is a twin from > p32. > Talking about the asu of p3221, it holds 3 chains, the chain of interest > assigned as 2 conformations related by a strict 2-fold. Since you believe > and have reduced to P3221, then you should restrain the two overall > strictly (since absolutely most of these atoms are not on special > positions) and not to refine the two 0.5 occupancies either, respecting the > crystallographic 2-fold. This is equivalent to perfect twin from p32 > theoretically (although refinement with twin mode in p32 and none-twin mode > in p3221 may give difference even large). If you refined and resulted in > occupancies away from 0.5, the symmetry was proven to be broken, which > supports P32 twinning. Lijun > > > Sent from my iPhone > > > > > > On Aug 27, 2021, at 6:56 AM, Oganesyan, Vaheh < > [email protected]> wrote: > > > How P3221 can be an option if it assumes chain on axis? I guess I’m > missing something, but per my belief only those sg will be possible for > which there is no axis going through the extra molecule. P1 sg looks the > only correct option here in my humble opinion. > Democracy (voting) depends on science. However, the reverse is not, > thankfully. > > Vaheh > > > > From: CCP4 bulletin board <[email protected]> On Behalf Of Peer Mittl > Sent: Friday, August 27, 2021 6:32 AM > To: [email protected] > Subject: Re: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold > axis? > > Dear Herman, > > The answer probably depends on the impact of the "extra" chain on the > sublattice. If there is no impact the "true" space group is P3221 with > one chain on the special position. If the swapping of the extra chain > influences the sublattice P32 (or C2 or P1, as pointed out by Kay) > twinned to P3221 might be the better description. > > All the best, > Peer > > On 27.08.2021 10:56, Schreuder, Herman /DE wrote: > > > > Dear Peer and Eleanor, > > > > This is indeed what I am suspecting: If the “twinning operator” in P32 > > puts 4 out of 5 protein chains on top of symmetry mates, is the “true” > > space group then P32, with 5 twinned chains, or P3221 with 4 normal > > chains and 1 chain on a special position? I would vote for the latter. > > > > Best, > > > > Herman > > > > *Von:* CCP4 bulletin board <[email protected]> *Im Auftrag von > > *Peer Mittl > > *Gesendet:* Freitag, 27. August 2021 10:17 > > *An:* [email protected] > > *Betreff:* Re: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis? > > > > Dear Eleanor, > > > > I indeed used r/tefmac for the refinement and it came up with the values > > HKL (a=0.56), KH-L (a=0.44). It would be interesting to see if a > > refinement in P3221 would come up with the same occupancies for the > > alternative conformations for the "extra" chain on the 2-fold axis. It > > seems as if the "well-ordered" chains (2 in P3221, 4 in P32) form a > > sublattice with P3221 symmetry and it's just the "extra" chain, which > > generates the twinning. > > > > All the best, > > Peer > > > > On 26.08.2021 18:09, Eleanor Dodson wrote: > > > Motto =mitti in predictive text! > > > > > > On Thu, 26 Aug 2021 at 16:52, Eleanor Dodson > > > <[email protected] <mailto:[email protected] > > <mailto:[email protected]%20%3cmailto:[email protected]>>> > > > wrote: > > > > > > Great, motto. I think you have nailed it! Did you use tefmac for > > > twinned refinement? And if so what did it suggest the twin > > > fraction is? > > > > > > On Thu, 26 Aug 2021 at 16:30, Peer Mittl <[email protected] > > <mailto:[email protected]%0b>> <mailto:[email protected] > > <mailto:[email protected]>>> wrote: > > > > > > Yes, the data indeed seems to be twinned and the tNCS has > > > masked the twinning statistics, which is why I haven't > > > considered it so far. > > > > > > I have not tried twinned refinement in C2 and P1 yet, but > > > refining 4 chains in P32 with twinning yields a difference ED > > > map that clearly indicates one (and just on!) orientation for > > > the 5th chain. Thank you all for your suggestions. > > > > > > Have a nice evening, > > > Peer > > > > > > -----"CCP4 bulletin board" <[email protected] > > <mailto:[email protected]%0b>> <mailto:[email protected] > > <mailto:[email protected]>>> schrieb: ----- > > > An: [email protected] <mailto:[email protected]> > > <mailto:[email protected] <mailto:[email protected]>> > > > Von: "Kay Diederichs" > > > Gesendet von: "CCP4 bulletin board" > > > Datum: 26.08.2021 16:41 > > > Betreff: Re: [ccp4bb] chain on 2-fold axis? > > > > > > Dear Peer, > > > > > > I suspect that the true spacegroup has lower symmetry than > > > P3221, and that there may be twinning masked by tNCS. > > > Subgroups of P3221 are C2 and P32 ( > > > > > > https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups > > < > https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups > > > > > > > < > https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups > > < > https://strucbio.biologie.unikonstanz.de/xdswiki/index.php/Space_group_determination#Subgroup_and_supergroup_relations_of_these_space_groups > >> > > > ) > > > and of course P1. > > > What I'd do is process the data, and solve (use the best chain > > > of the refined P3221 model for MR) and refine the structure in > > > these spacegroups. > > > Inspect the results: If P1 is clearly better than P32 and C2, > > > P1 is correct. > > > If C2 (P32) is clearly better than P32 (C2), then P1 should > > > give the same R-values as the better one; if so, P1 can be > > > discarded. > > > Try this with and without twin refinement - although it's hard > > > to compare R-values of non-twinned and twinned refinements. > > > > > > The automatic way to do this is with Zanuda. If you run that > > > locally, you can make refmac do twin refinement. > > > > > > For all resulting structures, I'd also feed the resulting > > > Fcalc (!) into pointless. That should reveal that the packing > > > is indeed close to P3221. > > > > > > Best wishes, > > > Kay > > > > > > > > > On Thu, 26 Aug 2021 11:54:06 +0200, Peer Mittl > > > <[email protected] <mailto:[email protected] > > <mailto:[email protected]%20%3cmailto:[email protected]>>> wrote: > > > > > > >Der CCP4 community, > > > > > > > >Is there a refinement program that can handle protein > > > monomers sitting > > > >on crystallographic 2-folds? > > > > > > > >This is probably a strange question but we have the following > > > situation. > > > >We have a 2.6 Ang datasets in SG P3221 (Rpim=4%, Isa=19.6) > > > and a clear > > > >molrep solution with 2 chains, albeit with tNCS (0/0/0.5) > > > that can be > > > >refined to around 27/33% Rfactor. According to Vm a third > > > chain could be > > > >present. So far so good, but there is clear difference ED for > > > a third > > > >chain sitting exactly on the 2-fold. Since the protein has a > > > peculiar > > > >shape, one can tell even its orientation. I can relax the > > > symmetry to > > > >P32 (or even P1) and place the missing chain with 50% > > > occupancy on the > > > >2-fold. This model can be refined, but I do not like this > > > work around, > > > >because the data is clearly P3221. > > > > > > > >Any hints on similar crystal pathologies and how they have > > > been handled > > > >would be helpful. > > > > > > > >All the best, > > > >Peer > > > > > > > > > > >######################################################################## > > > > > > > >To unsubscribe from the CCP4BB list, click the following link: > > > >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > > > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1>> > > > > > > > >This message was issued to members of > > > www.jiscmail.ac.uk/CCP4BB <http://www.jiscmail.ac.uk/CCP4BB> > > <http://www.jiscmail.ac.uk/CCP4BB <http://www.jiscmail.ac.uk/CCP4BB>>, > > > a mailing list hosted by www.jiscmail.ac.uk < > 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