Hi Nat and Phenix community, For high resolution structures, below 1.5 Å, is there a set of rules for best practices for refinement? I've been toying with different parameters and found that the weighting didn't make a huge difference, which is probably expected, with a 1.3 Å dataset. Do stereochemical/ADP weights have any effect? With weighting – Rwork 0.1573 Rfree 0.1806 RMS (bonds) 0.0073 RMS(angles) 1.468 Clashscore: 6.1 Ramachandran: 100 Ramachandran outliers: 0 Rotamer outliers: 0 CC ~0.94 Without weighting- Rwork: 0.1603 Rfree: 0.1840 RMS (bonds) 0.0049 RMS(angles) 1.272 Clashscore: 7.6 Ramachandran favored: 100 Ramachandran outliers: 0 Rotamer outliers: 0 CC ~0.94 For the particular data set I'm working with, I'm refining directly against anomalous data, more specifically against 12 Iodines. Refining the f' and f" has worked well, generating a beautiful density map. Refining against the model with hydrogens increased the Rfree/Rwork values by about 2 points. From what I've read previously I thought refining hydrogens in a non-"riding" model was only valid below ~1.0 Å. The previous phenix notes http://www.phenix-online.org/presentations/latest/pavel_phenix_refine.pdf , slide 23 has the combination of anisotropic B-factors with TLS refinement for atoms. This combination worked well for reducing the Rfree/Rwork values by a few points. For water, I assume that at this resolution there is a combination of anisotropic (big banana looking density) and isotropic waters and that one cannot simply rely entirely on the water_update option. Partial occupancy has been hit or miss, during one refinement it will appear that there is density for an alternative conformation. After adding the alternative conformation the density will disappear or show that no density should be there. In other instances refinement of the partial occupancies has worked well. The twin law has worked great, usually dropping Rfree/Rwork values by 5 points or so. I apologize if any of this is common knowledge and shows my relative inexperience when it comes to best practices for crystal refinement. Ryan Spencer University Of California, Irvine Nowick Research Group -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Pavel Afonine Sent: Friday, October 11, 2013 12:17 PM To: [email protected] Subject: Re: [phenixbb] Problem in reducing R-factor I'm *thinking* (not doing it right now) to make phenix.refine do something of this sort automatically if the resolution is really 1.3A (and not in fact say 1.5 or so but data file has a couple of reflections nearing 1.3A, which would tend people claim it is 1.3A resolution!) and r-factors are not too far to expected ones given *actual* data resolution. Of course "1.3A" in this case is going to be a range between 1 and 1.5A in terms of data quality. Pavel On 10/11/13 8:39 AM, Nathaniel Echols wrote:

What happens if you try refining protein atoms (but not waters) with individual anisotropic B-factors instead of TLS? It isn't guaranteed to work at 1.3Å, but it usually does (in my hands, anyway).

Also make sure you have explicit hydrogens added; this will have a significant effect at this resolution. And turn on the water update if you haven't already.

On Fri, Oct 11, 2013 at 6:19 AM, Singh, Bishal mailto:[email protected]> wrote:

Hi all, I am solving a crystal structure at 1.3Å. Completeness of the data-set and Figure of merit at this resolution are 99.5% and 0.62 respectively. Structure has two molecules per asymmetric unit. I used TLS parameters and NCS restraints. WXC_scale and WXU_scale are 0.50 and 1.0 respectively. Rwork and Rfree are 18.09 and 19.62 respectively. I tried with reducing WXC_scale and WXU scale to 0.25 and 0.30 respectively but it did not help. Please suggest me if I need to change some parameters. I shall be grateful for your help.

Thanking you, Bishal Singh Graduate student University of Heidelberg, Germany _______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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