[phenixbb] Discrepancy between R-factors from phenix.refine vs phenix Generate "Table 1"

Pavel Afonine pafonine at lbl.gov
Wed Jun 12 14:13:19 PDT 2013


just for completeness... Correctly selecting free-r flags is essential 
in case of twinning (phenix.refine does it by default). If flags were 
chosen without having that in mind then "R=0.24, Rfree=0.28" from your 
example may easily become "R=0.24, Rfree=0.34" once you switch to 
correctly generated flags. I've seen it more than once.


On 5/31/13 12:15 PM, Jon Schuermann wrote:
> Sorry if this is a little long, but I saw that more than one person 
> was having a similar problem.
> First, I agree with everything Nat has said, but I will explain a 
> little bit more. Twinning and/or pseudosymmetry can be a hairy issue 
> to deal with. In some cases, NCS can be a real pain. I have seen many 
> cases where just pseudosymmetry is present, but people assume it is 
> twinned as well. To be fair, it is very easy to come to that 
> conclusion, especially at the early stages. Let me just set the stage 
> with a hypothetical example...
> You collect a 2.5A dataset that processes well in P622 with an 
> Rsym=0.11 (overall). The intensity statistics look normal, which I 
> won't go into right now because it is complicated (even though Nat 
> eluded to it with the 'L-test'). You perform MR with a search model 
> that has 60% sequence identity and get a solution with 1 molecule in 
> the AU in P6522. The solvent content is 0.6. You run the solution 
> through rigid-body, followed by positional and B refinement with 
> simulated annealing. The resulting R=0.35, Rfree= 0.40. You look at 
> the maps and the density does not look good. You can see density for 
> most of the main-chain but almost all of the side-chain density is not 
> present. You go about trying to build in the density but the R/Rfree 
> do not drop much. What is your next step? Some would go to Se-Met or 
> heavy atom soaks to solve by SAD or MIR, but the results are the 
> same... Now What? Most people would say, its hexagonal where 
> merohedral twinning is possible so it must be twinned. You rescale the 
> data in P6, P312, P321, and even P3 (tetartohedral twinning) and the 
> Rsym is about the same in all the subgroups with P6 and P3 being the 
> lowest with Rsym=0.08. You perform MR in P65 and get a solution with 2 
> molecules in the AU. To 'check' if it is twinned, you run refinement 
> twice from the same starting model; first, twin refinement specifying 
> the 'twin law' (from XTRIAGE), and second regular refinement and then 
> compare the results. You think the results speak for themselves... 
> with the twin refinement, the R=0.24, Rfree=0.28 with the twin 
> fraction refining to 0.49 (nearly perfect twinning). The maps look 
> great! The regular refinement (without specifying the 'twin law') 
> refines to R=0.30, R-free=0.32 and the maps look better than P6522, 
> but not as good as the maps from the twin refinement. You think, 'This 
> has to be twinned!' But here is the kicker, IT'S NOT TWINNED!!! How 
> could this be???
> When you input the 'twin law' in refinement and the twin fraction 
> refines to >0.4, Phenix detwins using the proportionality rules. This 
> method uses the model in the detwinning process which WILL introduce 
> model bias in the maps, so they look great. Even if your model is NOT 
> correct, the maps will still look good, because of the model bias. For 
> this reason, you should calculate several 'omit maps' over different 
> regions of your structure and inspect them, or look at maps calculated 
> from refinement when you did NOT specify the 'twin law'. As for the 
> R-factor being lower, the calculated twin R-factor is usually lower 
> than the standard R-factor since the equations are NOT the same. So, a 
> drop in R-factor at this early stage is not necessarily conclusive 
> that your data is twinned. There are a couple of papers explaining 
> this (which Nat eluded to and I believe I reference below).
> I have seen many people get 'itchy finger' and try to use the twin 
> refinement too early. As Nat mentioned, you don't want to run it 
> unless you are at the end of model building and ready for deposition, 
> but the R-factor is still too high. Then it may be necessary to run 
> refinement with the 'twin law' included to see if the R-factor drops 
> 'significantly'.
> To summarize, if you suspect an issue with twinning and/or 
> pseudosymmetry since the maps don't look as good as they should for a 
> given resolution and the R-factor is too high, rescale/reprocess your 
> data in lower symmetry SG's and rerun MR. When you get a solution, 
> refine it WITHOUT specifying 'twin law' and look at the maps.
>     A)If they look much better than the solution in the higher 
> symmetry SG, continue your model building and refinement (WITHOUT 
> 'twin law') cycles in this lower symmetry SG until the model building 
> is complete and you would be ready to deposit the structure in the 
> PDB. If the R-factor is still too high, then run refinement including 
> the 'twin law' with the twin operator from Xtriage. If you see an 
> appreciable decrease in R-factor, then you probably have twinning. If 
> the R-factor dropped, but is still not low enough, then either your 
> model is still not completely correct or you have something else going 
> on as well (see end of B below).
>    B) If the maps still do not look any better than the higher 
> symmetry SG, try MR in another lower symmetry SG and repeat. I 
> sometimes go down to P1, if I have enough data. If the maps still look 
> bad (assuming you have a complete dataset and MR solution), then you 
> may not have a SG issue at all, but something else. I would look into 
> other issues including anisotropy, pseudo-translational symmetry, 
> order-disorder, missing molecules, etc. I will usually scroll through 
> the images to see if I see something obvious. Reprocess the data more 
> carefully looking for things like extra spots not predicted, or 
> predicted spots that aren't actually there, etc.
> Here are a few references to papers that explain some of these issues 
> more in depth with examples:
> Acta Crystallogr. (2008), D64, 99-107.
> Acta Crystallogr. (2012), D68, 1541-1548.
> Acta Crystallogr. (2006), D62, 83-95.
> I hope this helps some people...
> Jon
> -- 
> Jonathan P. Schuermann, Ph. D.
> Beamline Scientist, NE-CAT
> Argonne National Laboratory, 436E
> 9700 S. Cass Ave.
> Argonne, IL 60439
> Email:schuerjp at anl.gov
> Tel: (630) 252-0682
> On 05/30/2013 09:26 AM, Nathaniel Echols wrote:
>> On Thu, May 30, 2013 at 7:09 AM, Heather Condurso<condurso at bc.edu>  wrote:
>>> I have a similar issue.  My data is nearly perfectly twinned so Xtriage usually fails to find any twin law.  However when I solve the structure in the lower symmetry space group with twinning my maps look beautiful.  In the higher symmetry space group, only 3 of the 6 monomers fit into nice looking density.  I simply use generate table 1 and uncheck the box so it won't regenerate R factors.  The discrepancy between the R-factors is however much more significant than 2%.  From refine I get 18/22 and from table1 28/31.
>> That's actually a little surprising that Xtriage wouldn't work - would
>> you be willing to send us the data?  Keep in mind that if you have
>> perfect twinning and refine with the twin law, I think the maps will
>> look beautiful pretty much by definition, because of the model bias.
>> (Which doesn't mean that you did it wrong - the R-factors sound
>> realistic - you just need to be very careful and make lots of omit
>> maps.)
>> It helps to have actual examples when warning against the dangers of
>> twin refinement and model bias in general, so here's one (attached).
>> This map isn't beautiful, but it matches the model very closely,
>> including the complete lack of sidechains.  Another Phenix user got
>> this result by accident with a mostly-if-not-entirely incorrect MR
>> solution - fortunately the combination of missing sidechain density
>> and relatively high R-free (~40%) prompted him to email us.  I suspect
>> that if we searched the PDB we'd find an actual published example
>> (especially considering how many spurious ligands have been found
>> there).
>> (Sorry to sound like a broken record here, but this keeps coming up in
>> emails and workshops - perhaps we need better validation tools.)
>> -Nat
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