[phenixbb] XN-refinement

Johannes Schiebel johannes.schiebel2 at gmail.com
Fri Oct 14 07:50:37 PDT 2016

Dear Pavel.

Thank you very much for your answer and explanations. Actually, in the 
first place I already refined against neutron data (without X-ray) until 
convergence and the results are quite good. However, water orientations 
(in which we are particularly interested) are not easily discernible in 
the maps since the knowledge about water oxygen positions from the X-ray 
data are not explicitly implemented during refinement. Hence, we 
collected room-temperature X-ray data from a deuterated crystal grown in 
the same drop as the crystal used for neutron diffraction experiments. 
With the combined XN-data, it is astonishing how the R-free of both 
X-ray and neutron data drop significantly (by 2% for the neutron part) 
and how well the data agree. To account for the longer X-D/H bond 
lengths in nuclear density maps, we used the respective restraints for 
neutron data (Settings --> Advanced --> Model interpretation --> Use the 
nuclear distances for X-H/D).

Overall, I would really like to use the XN-refinement approach to 
improve the model in particular with respect to its water structure. 
Ultimately, we will anyhow deposit the neutron, X-ray and joint XN 
structures. Is it not possible at all to achieve coinciding coordinates 
and identical ADPs when using X and N at the same time?

The only alternative that I have not already used and would further 
improve the water structure in the model would be what you describe 
under 3. From what I read about it, however, it will be difficult to 
generate restraints also for water oxygens for which it would be 
particularly important in our case. Or is this possible in addition to 
the dihedral restraints for the protein? Thanks a lot!


Am 14.10.2016 um 16:09 schrieb Pavel Afonine:
> Hi Johannes,
> historically, in joint Xray+Neutron refinement first tried by Coppens 
> et al (1981) in small molecule world and later (1982) by Wlodawer & 
> Hen­drickson in bio-macromolecules, and way later implemented in 
> refinement tools such as nCNS (2009) and Phenix (2010), one single 
> model is refined against two data sets, Xray and neutron.
> The rationale to use two data sets, Xray and neutron, is to alleviate 
> the seemingly poor data-to-parameters ratio because H and D are used 
> explicitly in refinement using neutron data. This is all discussed in 
> great details in that 6 years old paper:
> https://www.phenix-online.org/papers/dz5209_reprint.pdf
> Turns out with a proper data / restraints weight the problem of 
> data-to-parameters amount is not as bad as it may appear, since by 
> varying the weight one can dose the amount of a priori knowledge 
> (restraints) as desired. This makes it possible to refine one model 
> against X-ray or neutron data individually without problems.
> Also, we know that X-H distances for X-ray and neutron are different 
> (X-ray are shorter a tiny bit). We also know that both data sets may 
> be collected at different temperatures (neutron at room, and X-ray at 
> cryo), which means B factors are going to be different, some rotamers 
> may be different, water structure may be slightly different, and so 
> on. All in all we really do need to have two structures to maximize 
> the use of available information. This is the new refinement paradigm 
> that's being implemented in phenix.refine and will become available at 
> some point.
> The best you can do right now is:
> 1) Get the best possible refined X-ray structure (refined against 
> x-ray data set alone);
> 2) Use refined x-ray structure as a starting point for neutron 
> refinement (add H,D as appropriate - trivially done using 
> phenix.ready_set or using Phenix GUI);
> 3) Refine structure from #2 against neutron data set alone. This will 
> make sure H and D have identical coordinates and B. If really needed 
> you can supply X-ray structure as a reference model - also trivially 
> done from the phenix.refine GUI.
> Let us know should you have questions or need help.
> Pavel
> On 10/13/16 23:06, Johannes Schiebel wrote:
>> Hi everyone,
>> I am currently working on an X-ray/neutron (XN) joint refinement 
>> using phenix.refine. As it should be, H/D coordinates and ADPs at 
>> exchangable sites are refined to equal values when using Phenix 
>> version 1.10.1-2155 and neutron data only. This is also stated in the 
>> paper describing the development of the method (Afonine et al. (2010) 
>> Joint X-ray and neutron refinement with phenix.refine. Acta 
>> Crystallogr D Biol Crystallogr 66, 1153-1163) as the default 
>> behavior: "Currently, phenix.refine maintains the H and D atoms at 
>> coinciding positions and constrains their ADPs to be equal to each 
>> other". However, when switching to XN-refinement using the same 
>> Phenix version, H and D atoms refine to different coordinates and 
>> ADPs, which should not be the case as it leads to unrealistic 
>> artifacts as can be seen from the deposited PDB 3X2P:
>> ATOM    186  H  AALA A  13       3.486 -18.200 -14.123  0.38 
>> 14.54           H
>> ATOM    187  D  BALA A  13       3.488 -18.195 -14.111  0.62 
>> 38.07           D
>> In this example, the D-occupancy is likely overestimated, while the 
>> H-occupancy is underestimated because the ADPs refine to very 
>> different values, which is chemically not reasonable.
>> Hence my question: How can I treat my data in a way that ADPs and 
>> coordinates refine to the same values at exchangeable H/D sites also 
>> for XN-refinement? Is there a specific keyword I am currently 
>> overlooking or do I have to use another Phenix version?
>> I would be really glad to receive your feedback. Thanks in advance!
>> Kind regards,
>> Johannes
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