Dear Emily, In this case, I was referring to the difference between 1065 obtained placing 4 copies of the monomer and 722 obtained placing 2 copies of the dimer. A difference of over 300 is substantial when we feel that an increase of 60 is enough to be reasonably convinced that a solution for placing one copy of a molecule is correct. I guess that this value of 60 would be my yardstick for “significant” in the sense of “big enough to matter”. Putting a number to “significant” in the sense of “bigger than random” is harder. There’s an argument that the random error in an LLG value is roughly equivalent to the square root of the LLG, which would still make this a significant difference. That argument could get much more complicated if you started to worry about things like the 2-dimer model just being a constrained version of the 4-monomer model because the monomers within the dimer are identical!
Not sure if that really helps! Randy ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > On 18 Sep 2017, at 17:39, Emilia C. Arturo (Emily) <ecgart...@gmail.com> > wrote: > > Hello Randy, > > I'm chiming in about the last sentence in your reply: > > > Finally, I would suspect that getting a significantly lower LLG for two > copies of a dimer means that the dimer in your structure is slightly > different from the dimer in the model. > > Will you please be more specific about what you consider "significantly > lower" (or higher) for LLG scores? And how does this difference translate to > differences among TFZ scores? I have wondered this especially when a Phaser > MR result from use of a smaller part (i.e. one domain, 64% of the sequence) > of a structure differs by a few units relative to a result from use of a > larger part (i.e. two domains, 90% of the sequence) of the protein; the > difference in my case was a top TFZ score of 42.5 using the one-domain search > model, but a lower TFZ score of 36.9 for the two-domain model. > > Even more interesting to me is when the TFZ scores vary by quite a bit more > between two phaser runs that used two different, but 100% sequence identical, > one-domain search models; in this case the top TFZ scores were 18.7 versus > 42.5, for the two models that differed only in the coordinate position of ~20 > out of 290 residues (a lid for the enzyme). > > Regards, > > Emily. > > > Best wishes, > > Randy Read > > ----- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +44 1223 336500 > Wellcome Trust/MRC Building Fax: +44 1223 336827 > Hills Road E-mail: > rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > > On 18 Sep 2017, at 16:24, Andrew Leslie <and...@mrc-lmb.cam.ac.uk> wrote: > > > > Regarding the warning message "The top solution from a TF rescoring did not > > pack”, I get this on all the PHASER jobs that I have run recently, but > > looking through the PHASER log file I cannot see any evidence for packing > > failure. > > > > It may be that the failure is buried in an obscure place in the very long > > log file, but if I search for “pack” then I get the output summarised > > below, all of which, apart from the ADVISORY, suggests to me that there is > > no problem at all with the packing. > > > > Perhaps someone on the PHASER team can cast light on this. > > > > Andrew > > > > > > Extracts from PHASER log file: > > > > > > TRANSLATION FUNCTIONS > > --------------------- > > > > Target Function: FAST LETF1 > > Translation Packing Function applied: top peak will pack > > Translation Packing Cutoff: 50% > > Sampling: 1.82 Angstroms > > > > > > =============================================== > > ---------------- > > PACKING FUNCTION > > ---------------- > > > > There are 4 solutions to pack > > Packing analysis > > 0% 100% > > |=====| DONE > > > > <!--SUMMARY_BEGIN--> > > Packing Table > > ------------- > > Solutions accepted if pairwise clashes less than 10 % of trace atoms > > #in #out Clash-% Symm TF-SET ROT TFpk# TF TFZ SpaceGroup > > 1 Top1 0.741 -- 1 1 1 325.94 26.03 P 1 > > 2 2 1.058 -- 1 2 1 293.29 24.34 P 1 > > 3 3 0.952 -- 1 3 1 245.70 21.88 P 1 > > 4 4 0.847 -- 1 4 1 211.49 20.11 P 1 > > > > 4 accepted of 4 solutions > > 4 pack of 4 accepted solutions > > > > ================================================== > > ------------------- > > TRANSLATION PACKING > > ------------------- > > > > Translation Packing Cutoff: 50% > > All solutions have been packed > > > > ================================================== > > Packing Fast Search Translations... > > 9871 peaks > > 500 peaks over 1049.74 checked for packing > > Translation peak 1 first to be kept > > Done > > > > <!--SUMMARY_BEGIN--> > > New Top Packing Fast Translation Function FSS = 2181.37 (TFZ=46.3) at > > Trial #1 > > <!--SUMMARY_END--> > > > > Top Peaks Without Clustering > > Select peaks over 67.5% of top (i.e. 0.675*(top-mean)+mean) > > There was 1 site over 67.5% of top > > 1 peak selected > > The sites over 67.5% are: > > # Frac X Frac Y Frac Z FSS Z-score > > 1 0.719 0.122 0.890 2181.4 46.35 > > > > Top 1 translations before clustering will be rescored > > Calculating Likelihood for TF SET #1 of 1 TRIAL #1 of 1 > > 0% 100% > > |==| DONE > > > > Packing LLG Translations: pass 1 of 11... > > 1 peaks > > No peaks over 1878.46 - no packing check > > Packing LLG Translations: pass 2 of 11... > > 1 peaks > > 1 peaks over 1878.46 checked for packing > > Translation peak 1 first to be kept > > Done > > Exit: found a peak that packs > > > > -================================================== > > > > *** AND THEN *** > > ------------------------------------------------------------------------------------------ > > Advisory: The top solution from a TF rescoring did not pack > > ------------------------------------------------------------------------------------------ > > > > > > -================================================== > > - > > > >> On 18 Sep 2017, at 15:06, Eleanor Dodson <eleanor.dod...@york.ac.uk> wrote: > >> > >> You need to provide a bit more information. > >> > >> First of all about the data processing.. > >> > >> Is the space group correct? > >> ways of being misled are: > >> Non-crystallographic translations with a shift of ~0.5 along an axis - say > >> a. This will generate absences in the odd h 0 0 reflections and can make > >> the space group appear to be P 21 21 21 whilst it is really P 2 21 21.. > >> > >> Perfect twinning can have the same effect. In an orthorhombix space group > >> this can usually only occur if two axes have approximately the same > >> length, but the data processing stats can indicate if that is the case. > >> > >> Then - re PHASER. The packing rejection criteria may be set too severely - > >> that seems the case for your solution. > >> > >> Best check on any MR solution is: does it refine - give it 20 cycles of > >> mindless refinement and see if the R and FreeR go down. > >> > >> Then look at the maps and see if there are obvious corrections to be made.. > >> > >> Eleanor > >> > >> On 18 September 2017 at 14:59, Satvik Kumar <kumarsatvi...@gmail.com> > >> wrote: > >> Dear Crystallographers, > >> > >> I am trying to solve a structure in the space group P212121. Based on > >> Matthews coefficient, there are 4 molecules in the asymmetric unit. > >> > >> Based on my limited reading about using of Phaser, I understand that a > >> single chain should be used as search model even though many copies are > >> present in asymmetric unit. Am I correct? > >> > >> So when I use a single chain as search model and ask Phaser to search for > >> 4 molecules, Phaser identifies a single solution with a warning "The top > >> solution from a TF rescoring did not pack" and a warning "Search request > >> requires more scattering than defined in composition. Composition > >> increased to accommodate search components". But the final values reported > >> "PAK=2 LLG=1065 TFZ==22.6" indicate that phaser has solved the problem. > >> > >> Can anyone please explain the meaning of the warning. > >> > >> When I inspect the arrangement of the chains (attachment), I observe > >> minimal contact between the chains and a large cavity in the center. Can a > >> crystal form this way? > >> > >> I have also tried using the dimer as search model and asking phaser to > >> search for 2 molecules. Even in this case, Phaser finds a single solution > >> but the warning and the advisory still appear as before. The numbers > >> reported reduce a bit to "PAK=1 LLG=722 TFZ==29.2". > >> > >> Please help me in understanding these results. > >> > >> Thanks, > >> Satvik > >> > >> > >> > > > > > > -- > "Study as if you were going to live forever; live as if you were going to die > tomorrow." - Maria Mitchell > > "I'm not afraid of storms for I'm learning to sail my ship." - Louisa May > Alcott >
