Dear Petr,
I agree with Tom that there is no need to release the NCS in the final rounds
of refinement. You just end up throwing away good work. Perhaps you can tweak
the NCS a bit to try to get better results (even though your current results
sound fine). It's difficult to set up the right restraint weigths for NCS
especially if the NCS is not equally convincing in all parts of the structure,
it can be a lengthy trial-and-error process. The latest version of Refmac has
local NCS restraints which works a bit differently and does all the work for
you. Buster-TNT has the something similar (for quite a while actually). Both
work very well.
I always add riding hydrogens, even at low resolution. It doesn't add (m)any
parameters and the debumping effect can help your Ramachandran score. I have
not seen it backfire often. WHAT_CHECK will give you a global score of your
Rmachandran plot. It will tell you if you have improved your Ramachandran plot
even when the percentage outliers stays the same.
Cheers,
Robbie P. Joosten
Netherlands Cancer Institute
----------------------------------------
> Date: Fri, 27 Aug 2010 09:38:01 -0400
> From: [email protected]
> Subject: Re: [ccp4bb] Low-resolution structure refinement with Refmac
> To: [email protected]
>
> I would agree on both points- as to the NCS, there was a very useful study by
> Kleywegt
> of deposited structures that that released NCS at resolutions that didn't
> justify it-
> maybe
> G J Kleywegt, "Use of non-crystallographic symmetry in protein structure
> refinement", Acta
> Crystallographica, D52, 842-857 (1996).
> or
> G J Kleywegt & T A Jones, "Where freedom is given, liberties are taken",
> Structure, 3,
> 535-540 (1995).
>
> As to outliers, I understand adding (riding) hydrogens can improve the
> Ramachandran,
> but like enforcing dihedrals this somewhat invalidates the use of the
> Ramachandran,
> since it's mainly the clashes between hydrogens that define the ramachandran
> permitted area. I would not recommend adding H's at this resolution.
>
> If the outlier is well defined in the density, it is probably a real outlier;
> If as you say poorly defined, the data is not strong enough to determine the
> rotamer so it drifts out, and the rama plot is correctly indicating this.
>
> Ed
>
> Thomas Womack wrote:
>> On 27 Aug 2010, at 10:55, Petr Kolenko wrote:
>>
>>> Dear crystallographers,
>>>
>>> I have a structure at 3.3A resolution, 16 identical chains in AU,
>>> merohedral twinning present. I started to refine using NCS restraints with
>>> chain A as a reference chain. Current Rwork/Rfree is 21/25. There is almost
>>> nothing to refine manually in whole structure now. But, refinement without
>>> NCS restraints results in Rwork/Rfree of about 17/28. What should I do? Or
>>> is it possible to deposit the structure refined using NCS restraints in
>>> final refinement?
>>
>> This seems like a really well-done NCS refinement; using the multi-fold NCS
>> is allowing you to get what is an excellent Rfree and Rwork-Rfree gap for
>> 3.3A data. Definitely deposit the NCS-restrained version; refining without
>> NCS restraints just increases the number of parameters by a factor sixteen
>> and spends most of those on fitting noise.
>>
>> 1% Ramachandran outliers at 3.3A also seems entirely reasonable.
>>
>> Tom
>>
