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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex Lee Sent: Friday, April 14, 2017 11:50 AM To: CCP4BB@JISCMAIL.AC.UK 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 <eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> 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 <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>] Sent: Thursday, April 13, 2017 11:32 PM To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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:alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>] Sent: Thursday, April 13, 2017 9:08 PM To: Keller, Jacob <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 <kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>> 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). D73<http://journals.iucr.org/d/contents/backissues.html>, 22-31 https://doi.org/10.1107/S2059798316019318 Jacob From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Eleanor Dodson Sent: Thursday, April 13, 2017 3:11 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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 <r.joos...@nki.nl<mailto:r.joos...@nki.nl>> 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<mailto:alexlee198...@gmail.com> Verzonden: donderdag 13 april 2017 19:19 Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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.
