Dear Peer, I must admit that from the beginning I was not happy to have 5 chains “twinned”, while in fact only one chain is “twinned”, e.g. on a special position. However, a twin fraction different from 0.5 suggested the existence of twin domains and not a random distribution of A and B conformations.
On the other hand, given that only 20% of the asymmetric unit is twinned, with tNCS present and imperfect experimental data, the twin fraction obtained might not be very accurate and a twin fraction of 0.5 could be within the error margin. The low Rfactors you obtain without twinning also point into this direction. What I would do is have a critical look at the crystal packing to see if there are reasons why larger twin domains with the rogue chain only in one conformation would form. E.g. are there direct crystallographic contacts between these disordered that may force the disordered chain next to a chain in the “A” conformation to adopt the A conformation as well. Or is the disordered chain enclosed in the 4 non-disordered chains, with only crystal contacts between the non-disordered chains? If there is no compelling reason why neighboring disordered chains would adopt the same conformation, I would assume statistical disorder and not twinning. My 2 cts, Herman Von: mi...@bioc.uzh.ch <mi...@bioc.uzh.ch> Gesendet: Dienstag, 31. August 2021 17:46 An: Schreuder, Herman /DE <herman.schreu...@sanofi.com> Cc: CCP4BB@JISCMAIL.AC.UK Betreff: Antwort: [ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Antwort: Re: [ccp4bb] chain on 2-fold axis? 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" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> schrieb: ----- An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 <lijunli...@gmail.com<mailto:lijunli...@gmail.com>> Gesendet: Freitag, 27. August 2021 15:57 An: Schreuder, Herman /DE <herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> Cc: ccp4bb@jiscmail.ac.uk<mailto:ccp4bb@jiscmail.ac.uk> 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 <herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> 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 <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Im Auftrag von Lijun Liu Gesendet: Freitag, 27. August 2021 14:22 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 <vaheh.oganes...@astrazeneca.com<mailto:vaheh.oganes...@astrazeneca.com>> 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 <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> On Behalf Of Peer Mittl Sent: Friday, August 27, 2021 6:32 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 > <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> *Im Auftrag von > *Peer Mittl > *Gesendet:* Freitag, 27. August 2021 10:17 > *An:* CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > *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 > > <eleanor.dod...@york.ac.uk <mailto:eleanor.dod...@york.ac.uk <mailto:eleanor.dod...@york.ac.uk%20%3cmailto:eleanor.dod...@york.ac.uk%20%0b>> <mailto:eleanor.dod...@york.ac.uk%20%3cmailto:eleanor.dod...@york.ac.uk>>> > 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 <mi...@bioc.uzh.ch <mailto:mi...@bioc.uzh.ch%0b>> <mailto:mi...@bioc.uzh.ch%0b>> <mailto:mi...@bioc.uzh.ch <mailto:mi...@bioc.uzh.ch%20%0b>> <mailto:mi...@bioc.uzh.ch>>> 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" <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK%0b>> <mailto:CCP4BB@JISCMAIL.AC.UK%0b>> <mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK%20%0b>> <mailto:CCP4BB@JISCMAIL.AC.UK>>> schrieb: ----- > > An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > > <mailto:CCP4BB@JISCMAIL.AC.UK> > <mailto:CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK%20%3cmailto:CCP4BB@JISCMAIL.AC.UK>>> > > 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>> > > > <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 > > <mi...@bioc.uzh.ch <mailto:mi...@bioc.uzh.ch <mailto:mi...@bioc.uzh.ch%20%3cmailto:mi...@bioc.uzh.ch%20%0b>> <mailto:mi...@bioc.uzh.ch%20%3cmailto:mi...@bioc.uzh.ch>>> 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>> > > <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 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<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>>> > > > -- > **************************** > Peer Mittl, PD Dr. > Biochemisches Institut > Universität Zürich > Room 44M03 > Winterthurer Strasse 190 > CH-8057 Zürich > > Tel. +41-(0)44-6356559 > Mobile +41-(0)76-2776566 > Fax. +41-(0)44-6356805 > Mail mi...@bioc.uzh.ch<mailto:mi...@bioc.uzh.ch> <mailto:mi...@bioc.uzh.ch> > > ######################################################################## > > 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>> 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