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>> Generally, the very same reason that makes it difficult to find an independent test set under a regime of high-order and genuine NCS implies that you don't need it! This type of NCS couples amplitudes and phases so inextricably (unlike crystallographic symmetry, that relates amplitudes to amplitudes and phases to phases without creating any interaction between amplitudes and phases) that it essentially protects you against phase bias: you can't get good agreement with the measured amplitudes without at the same time being forced towards phases that are a good approximation to the correct phases. This is where the power of this type of NCS comes from in enabling you to iteratively improve poor starting maps into stunnning ones, if you have that NCS accurately described. If this is true, why don't we pretend that our e.g. P622 crystals are P1 with 24-fold NCS, thereby coupling phases and amplitudes, and solving phase bias issues? Where does this phase-amplitude coupling come from? JPK On Mon, Mar 07, 2016 at 12:53:14PM +0100, Robbie Joosten wrote: > Hi Zheng, > > Assuming your choice of test set is not terrible, the small gap could be fine. That is, if the symmetry is really P1 and not higher (check this some more, 6-fold NCS in P1 makes people suspicious). With high NCS is becomes increasingly difficult to get an independent test set, this is why with viral capsids some authors don't even bother using a test set. > That said, a small R-factor gap may mean that your model is not done yet. At 1.6A you can try a lot of things such as anisotropic B-factors instead of TLS (Ethan Merritt wrote a nice paper about this, you can also try PDB_REDO to use the Hamilton test he proposes) and you can build a lot of non-protein, so go through the difference density a bit more thoroughly than normal. The average R/R-free at this resolution is 18.0/21.2 (n = 2321) in the PDB and 16.7/19.5 in PDB_REDO, so the average gap is quite a bit larger. The numbers are similar if you only take the P1 cases (n =110). > > Cheers, > Robbie > > > > > > -----Original Message----- > > From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf > > Of Zheng Zhou > > Sent: Monday, March 07, 2016 11:23 > > To: [log in to unmask] > > Subject: [ccp4bb] Rwork Rfree gap differences > > > > Dear all, > > > > I have sent the question to the buster discussion board two weeks ago. > > I think I'd better consult this problem on the active ccp4 discussion board. > > Sorry for a bit off topic. > > > > I recently solved a P1 crystal structure, 6 fold NCS at 1.6 angstrom resolution. > > I used lattice symmetry to generate test set in phenix. > > The phenix.refine gave me Rwork/Rfree = 0.1828/0.2081. There are > > pieces of positive and negative density for the compounds. > > > > So I tried buster refinement with autoncs, TLS. The density for the > > compounds are much improved, no obvious positive density. However, > > It generated Rwork/Rfree = 0.1801/0.1912. I also find similar R > > factor examples at the similar resolution in the buster wiki: > > > > https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExampl > > e4 > > chawaterNCS > > > > Would the small gap between Rwrok and Rfree be an issue? I have not > > deposited buster refined PDB yet, would this acceptable? Any > > reference to help if any reviewer questions about the small gap? > > > > Thanks for your advice in advance. > > > > Best, > > > > Zheng