Dear all
Thanks for the discussion and advice.
Finally the small molecule geometry issue was solved by using a new
library file generated after MoE energy minimization.
The Rfree/Rwork gap was improved using anisotropic ADP refinement
Rwork = 0.154
Rfree =0.191
Bonds RMSD 0.013
Angles RMSD 1.115
Best,
Zheng
On Thu, Mar 10, 2016 at 1:52 PM, Robbie Joosten
<[log in to unmask]> wrote:
> Hi Zheng,
>
> It looks like anisotropic ADPs don't help at all here so I wouldn't use
> them. Since this is the CCP4bb, you should also try Refmac for refinement.
> While you are at it, please also try PDB_REDO. Among other things, it tries
> to automatically optimise your refinement strategy for Refmac.
> In terms for making restraints, Grade from Global Phasing does a great job.
> I also use acedrg from CCP4 a lot.
>
> Cheers,
> Robbie
>
> Sent with my Windows Phone
> ________________________________
> Van: Zheng Zhou
> Verzonden: 10-3-2016 04:37
> Aan: [log in to unmask]
> Onderwerp: [ccp4bb] update on weight optimization Re: [ccp4bb] Rwork Rfree
> gap differences
>
> Dear all
>
> Thanks for your help.
>
> I attache the self rotation function from molrep. 6 fold NCS is
> visible. I could not find log file for
> phenix.reflection_file_converter
> --use-lattice-symmetry-in-r-free-flag-generation. How do I make sure
> the test set are chosen considering the NCS?
>
> I did anisotropic ADP refinement in phenix as suggested by Robbie
>
> Original Run TLS+isotropic ADP
> Rwork 0.1828
> Rfree 0.2081
> RMSD bonds 0.006
> RMSD angles 0.913
> comments: bits of positive and negative density at the compound position
>
> Run 1, anisotropic ADP
> Rwork 0.1674
> Rfree 0.2067
> RMSD bonds 0.006
> RMSD angles 0.869
> comments: still has positive and negative density at the compound
> position, but I re-modified some waters and loops, use this PDB as
> input for following refinements.
>
> Run 2, TLS+isotropic ADP+X-ray/stereochemistry weight optimization
> Rwork 0.1818
> Rfree 0.2074
> RMSD bonds 0.017
> RMSD angles 1.563
> comments: Compound density improved. Bond angle deviations are mostly
> at the compound.
>
> Run 3, anisotropic ADP+X-ray/stereochemistry weight optimization
> Rwork 0.1674
> Rfree 0.2056
> RMSD bonds 0.014
> RMSD angles 1.286
> comments: compound density is acceptable. Bond angle deviation are reduced.
>
> Run 4, TLS on protein+anisotropic ADP on
> compound+X-ray/stereochemistry weight optimization
> Rwork 0.1921
> Rfree 0.2135
> RMSD bonds 0.015
> RMSD angles 1.432
> comments: I had not done separate TLS and ADP before. Surprised to see
> it made the refinement statistics worse .
>
> Question: 1) At 1.6 angstrom resolution, the anisotropic ADP
> refinement is not justified as the Rfree did not decrease
> significantly. Is my understanding correct?
>
> 2) It looks like the weight optimization improves the compound
> density. Could the cif file be the problem? I input SMILES to phenix
> elbow AM1 to generate the cif file.
>
> 3) For Rfree set, should I force the index to P6 regardless of Rsym,
> generate test set and expand to P1? The Run 3 could be deposited. But
> Rfree is not improved comparing with the TLS refinement+isotropic ADP.
>
> Thanks for your time and reply in adance. I probably should send this
> to phenix board. But my original question get answers from CCP4 board.
>
> Best,
> Zheng (Joe) Zhou
>
> On Mon, Mar 7, 2016 at 8:26 PM, Gerard Bricogne <[log in to unmask]>
> wrote:
>> Dear Zheng,
>>
>> Robbie beat me to it, and his reply is very thorough.
>>
>> First of all, apologies for leaving your enquiry unanswered for
>> the whole of last week: we were all commandeered into reciprocal space
>> during that time. Claus will shortly be replying to your TLS question
>> on buster-discuss.
>>
>> I was going to ask you for a little more information on what you
>> mean by "I used lattice symmetry to generate test set in phenix." Do
>> you mean that your P1 lattice is nearly hexagonal? Or that you used a
>> feature that tries to assign free reflections as closely related by a
>> 6-fold symmetry axis (that you would have identified through a self-
>> rotation function) as possible?
>>
>> 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.
>>
>> In other words: IF your characterisation of your NCS and of your
>> space group are correct, don't worry about the small Rwork-Rfree gap:
>> you are in a situation that is far away from the typical one at that
>> resolution, and the statistics for structures of similar resolution,
>> most of which will not have NCS or not of such a high order, should
>> not intimidate you.
>>
>> If the difference density for the ligands was better with BUSTER,
>> was mostly consistent with the NCS without that having been imposed on
>> it, and was interpretable and refinable, then that would be quite a
>> bit of evidence that your result is correct.
>>
>>
>> With best wishes,
>>
>> Gerard.
>>
>> --
>> 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?AutoBusterExample4
>>> > 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
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