maybe attach your data processing log file.. e On 14 April 2017 at 20:10, Eleanor Dodson <[email protected]> wrote:
> That twin factor list means the apparent crystal symmetry must be P6/mmm. > > You say you only have 2 molecules in the asymmetric unit of P32,therefor > there must only be one in SGs P32 21 P32 12 > > So I dont understand why you have PHASER results like this: > > SOLU SET RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 > LLG=350 TFZ==20.5.. > > > Why so many TFZ here - is that achieved after refinement or something? > > > Eleanor > > > And what does the twinning analysis suggest? > > > > > On 14 April 2017 at 17:42, Keller, Jacob <[email protected]> wrote: > >> As I mentioned off-list, it would be helpful to know how many types of >> search models you are searching with—how many different molecules are in >> the complex? It’s hard to interpret MR results otherwise. >> >> >> >> Also, since the higher-symmetry SG works in MR, you should try to refine >> the model in that SG, with only two twin domains, refining twin fraction. I >> can guarantee that a good reviewer will have you do this (if not, then not >> a “good reviewer.”) >> >> >> >> JPK >> >> >> >> *From:* CCP4 bulletin board [mailto:[email protected]] *On Behalf Of >> *Alex Lee >> *Sent:* Friday, April 14, 2017 11:50 AM >> >> *To:* [email protected] >> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree >> surprisingly down >> >> >> >> Thanks Eleanor, I tried MR for P32 21 and P32 12. >> >> SG P3221: SOLU SET RFZ=5.3 TFZ=8.8 PAK=0 LLG=121 TFZ==11.2 LLG=944 >> TFZ==29.2 PAK=0 LLG=944 TFZ==29.2 >> >> SOLU SPAC P 32 2 1 >> >> >> >> SG P3212: >> >> Solution #1 annotation (history): >> >> >> >> SOLU SET RFZ=4.4 TFZ=7.7 PAK=0 LLG=55 TFZ==9.6 LLG=350 TFZ==20.5 PAK=0 >> LLG=350 TFZ==20.5 >> >> >> >> SOLU SPAC P 32 1 2 >> >> >> >> SG P32 >> >> SOLU SET RFZ=7.4 TFZ=10.4 PAK=0 LLG=187 TFZ==10.7 RF++ TFZ=17.0 PAK=0 >> LLG=436 TFZ==17.8 LLG=1715 TFZ==34.3 PAK=0 >> >> LLG=1715 TFZ==34.3 >> >> SOLU SPAC P 32 >> >> >> >> Based on TFZ and LLG, the P32 seems to be best. But I'll also try to refine >> and build P32 2 1 latter >> >> >> >> On Fri, Apr 14, 2017 at 4:32 AM, Eleanor Dodson < >> [email protected]> wrote: >> >> First - four way twinning is possible but pretty rare for macromolecules >> >> >> >> Pointless gives a very useful table of the CC agreement for each possible >> symmetry operator individually. >> >> In this case with only two molecules in the asymmetric unit you you could >> only have a higher symmetry SG as >> >> P32 21 P32 12 or P64 >> >> >> >> These would require as symmetry operators - >> >> P32 21 - a three fold and a two fold k h -l >> >> P32 12 - a three fold and a two fold -k -h -l >> >> >> >> P64 - a six fold >> >> >> >> If the scores for one set are better than the others you probably have >> that SG >> >> >> >> However high degrees of twinning can disguise the symmetry scores of >> course.. >> >> >> >> >> >> >> >> On 14 April 2017 at 04:46, Keller, Jacob <[email protected]> >> wrote: >> >> Try MR with one copy in all space groups of PG 321/312 using Phaser. >> Going from PG 3 to PG 32 should halve the number of copies per ASU. You may >> have to re-process your data in the higher point group to do this. >> >> >> >> Or you might actually have a tetartohedral twin, but just try with the >> higher-symmetry point group first, see what happens. >> >> >> >> JPK >> >> >> >> *From:* Alex Lee [mailto:[email protected]] >> *Sent:* Thursday, April 13, 2017 11:32 PM >> >> >> *To:* Keller, Jacob <[email protected]> >> *Cc:* [email protected] >> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree >> surprisingly down >> >> >> >> Hi Keller, >> >> >> >> Thanks for the suggestions! I only have two copies in ASU at SG P32. >> Zanuda also suggests P32 is the best SG. >> >> >> >> On Thu, Apr 13, 2017 at 8:12 PM, Keller, Jacob <[email protected]> >> wrote: >> >> Yes, this was my case exactly—it looks like there are two pairs of >> coupled twin domains: a,c and b,d. Assuming you have multiple copies of >> your model in the same ASU, try doing MR in higher symmetry space groups of >> point group 312 or 321, like P3212 etc. There is this handy page with all >> the space groups and their possible twin operators: >> http://www.ccp4.ac.uk/html/twinning.html. >> >> >> >> The twin fractions indicate a high twin fraction—~46% if actually >> hemihedral! >> >> >> >> Also take a look at the paper I referenced for more info. I can send you >> a .pdf if you need me to. >> >> >> >> Please let me know how it works out—I am interested in these types of >> things! >> >> >> >> JPK >> >> >> >> *From:* Alex Lee [mailto:[email protected]] >> *Sent:* Thursday, April 13, 2017 9:08 PM >> *To:* Keller, Jacob <[email protected]> >> *Cc:* [email protected] >> >> >> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree >> surprisingly down >> >> >> >> Hi Keller, >> >> >> >> I do not how to check twin fraction after Refmac (I guess it's somewhere >> in log file). From the log file it seems I have four twin domain: >> >> Twin operators with estimated twin fractions **** >> >> >> >> Twin operator: H, K, L: Fraction = 0.275; Equivalent operators: K, -H-K, >> L; -H-K, H, L >> >> Twin operator: -K, -H, -L: Fraction = 0.228; Equivalent operators: -H, H+K, >> -L; H+K, -K, -L >> >> Twin operator: K, H, -L: Fraction = 0.270; Equivalent operators: H, -H-K, >> -L; -H-K, K, -L >> >> Twin operator: -H, -K, L: Fraction = 0.228; Equivalent operators: -K, H+K, >> L; H+K, -H, L >> >> >> >> On Thu, Apr 13, 2017 at 4:36 PM, Keller, Jacob <[email protected]> >> wrote: >> >> What was the refined twin fraction after Refmac? It’s much more accurate >> than initial tests. Also, how many twin domains do you have? If you have >> many, it might be a higher space group but with less twinning. I recently >> had a case in which apparent tetartohedral (four-domain) twinning in P32 >> was really hemihedral (two-domain) twinning in P3212: >> >> >> >> *Acta Cryst. <http://journals.iucr.org/d>* (2017). D*73* >> <http://journals.iucr.org/d/contents/backissues.html>, 22-31 >> https://doi.org/10.1107/S2059798316019318 >> >> >> >> Jacob >> >> >> >> *From:* CCP4 bulletin board [mailto:[email protected]] *On Behalf Of >> *Eleanor Dodson >> *Sent:* Thursday, April 13, 2017 3:11 PM >> *To:* [email protected] >> *Subject:* Re: [ccp4bb] Refmac5 twin refinement pushing Rfree >> surprisingly down >> >> >> >> Twin refinement cannot be compared directly to untwinned - the R factors >> are between different parameters - without twinning it is assumed you have >> an amplitude obtained more or less from sqrt(I But for a twinned data set >> that I is actually [ I1 + twin_factor I2 ] so the amplitude is not really >> correct and twinned refinement will give a much better estimate. >> >> >> >> However you need to be careful that you have assigned the same FreeR flag >> to reflection pair related by the twin law. The modern program in the CCP4 >> data reduction pipeline looks after this pretty automatically - all >> possible symmetry equivalents are assigned the same FreeR but older >> software did not do this.. >> >> >> >> You can check it by looking at some twin equivalents - in SG P32 these >> could be h k l and -h, -k, l or h k l and k h -l or h k l and -k, -h, -l . >> >> >> >> Ideally they all should have the same Free R flag.. >> >> >> >> Eleanor >> >> >> >> PS - the acid test is: Do the maps look better? >> >> >> >> E >> >> >> >> >> >> On 13 April 2017 at 19:52, Robbie Joosten <[email protected]> wrote: >> >> Hi Alex, >> >> >> >> You are not giving the number after refinement without the twin >> refinement. Nevertheless, R-free drops like this are not unheard of. You >> should check your Refmac log file, it would warn you of potential space >> group errors. Refmac will also give you a refined estimate of the twin >> fraction. >> >> >> >> Cheers, >> >> Robbie >> >> >> >> Sent from my Windows 10 phone >> >> >> >> *Van: *Alex Lee <[email protected]> >> *Verzonden: *donderdag 13 april 2017 19:19 >> *Aan: *[email protected] >> *Onderwerp: *[ccp4bb] Refmac5 twin refinement pushing Rfree surprisingly >> down >> >> >> >> Dear All, >> >> >> >> I have a protein/dna complex crystal and data collected at 3A and another >> set at 2.8A, space group P32. L test shows twinning (fraction around 0.11). >> The structure solved by MR and model building of the complex finish (no >> solvent built yet, I do not think it's good to build solvent in such low >> resolution data). >> >> >> >> I did Refmac5 to refine my structure (restraint refinement) with or >> without twinning, to my surprise, the Rfree drops a lot after twin >> refinement of two data sets. Summary below: >> >> >> >> 2.8A dataset: before twin refine 34%, 29%; after twin refine:24%, 19% >> >> 3A dataset: before twin refine 30%;26%; after refine 25%, 18% >> >> >> >> I know that a lot of threads in CCP4bb talking about Rfree after twin >> refine and Rfree without twin refine can not compare directly. By drop R >> free this much by twin refine, it gives me a feeling of too good to be true >> (at such low resolution with such good Rfree, maybe overrefined a lot?), >> but from the density map after twin refine, it does seem better than no >> twin refine map. >> >> >> >> I do not know if reviewers are going to challenge this part. >> >> >> >> Any input is appreciated. >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> > >
