If real-space refinement was part of refinement then I wonder why you still have rotamer outliers. Their amount should be way smaller (close to 0 at this resolution, if not precisely zero!), so there must be something else that isn't quite right, which is hard to tell without looking at the files. Pavel On 7/28/13 2:33 PM, Wei Shi wrote:

Hi Dr Echols, Thank you for the suggestions. I ran the refinement again with default settings + Rigid body+ Simulated annealing (Cartesian) + Optimize X-ray/stereochemistry weight. And the statistics improved! See below: R-work=0.2348, R-free=0.2855, Bonds=0.003, Angles=0.919. Ramachandran outliers: 2.5%, Ramachandran favored: 88.1%. Rotamer outliers: 10%, c-beta outliers: 0 clashscore: 9.06, overall score: 2.84

Now, I am wondering whether there is any suggestion about what to try and check next to further refine the structure. Thank you so much!

Best, Wei

On Sun, Jul 28, 2013 at 4:00 PM, Nathaniel Echols mailto:[email protected]> wrote:

On Sun, Jul 28, 2013 at 12:44 PM, Wei Shi mailto:[email protected]> wrote: > I have a refined structure with phenix. The resolution=3.2 Å. After > refinement, the R-work=0.2186, R-free=0.2864, Bonds=0.010, Angles=1.515. > > Ramachandran outliers: 4.8%, Ramachandran favored: 80.8%. > > Rotamer outliers: 14.5%, c-beta outliers: 2 > > clashscore: 16.28, overall score: 3.32 > > To fix the Ramanchandran outliers, in coot, by changing the phi-psi of the > outliers, I could manage to move most of them to the allowed positions. But, > after another refinement in phenix, those outliers come back. > > I am wondering whether any of you have any suggestions for me on how to fix > these outliers.

Turn on X-ray/stereochemistry weight optimization (for non-GUI users, optimize_xyz_weight=True). That usually fixes the problem.

-Nat _______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Hi Wei,

Thank you guys so much for the suggestions! I don't know why I still have rotamer outliers after real-space refinement. I am wondering what might go wrong and what I should check in this case?

which Phenix version you use? Anyway, sounds like you don't have rotamer outliers as statistics below indicates.

At the same time, I fixed the Ramanchandran outliers and rotamer outliers in Coot, and did another refinement with default setting + NCS restraints + secondary structure restraints + optimize X-ray/ stereochemistry + optimize X-ray/ ADP. Are these refinement settings look reasonable to you? Below are the results: R-work: 0.2542, R-free: 0.2857, Angles: 1, Bonds: 0.003. Ramachandran outliers: 0.4% (Goal: <0.2%), Ramachandran favored: 92.6% (Goal: >98%). Rotamer outliers: 0 % (Goal: 1%), c-beta outliers: 0 (Goal: 0) clashscore: 6.92, overall score: 1.85.

Looks ok to me.

But, when I opened the results in coot, do Ramanchandran plot, and I couldn't see any Ramanchandran outliers.

This is because Coot and Phenix use slightly different Ramachandran plot libraries. I guess Paul Emsley or/and Jeff can offer more comments on this. Pavel

On Sun, Jul 28, 2013 at 2:33 PM, Wei Shi wrote:

Thank you for the suggestions. I ran the refinement again with default settings + Rigid body+ Simulated annealing (Cartesian) + Optimize X-ray/stereochemistry weight. And the statistics improved! See below: R-work=0.2348, R-free=0.2855, Bonds=0.003, Angles=0.919. Ramachandran outliers: 2.5%, Ramachandran favored: 88.1%. Rotamer outliers: 10%, c-beta outliers: 0 clashscore: 9.06, overall score: 2.84

Now, I am wondering whether there is any suggestion about what to try and check next to further refine the structure. Thank you so much!

To start with, don't run rigid-body refinement or simulated annealing in future refinements. They're both very powerful tools for improving structures which fit the data poorly, but once the model is mostly correct they just take forever with little benefit (and in the case of annealing, sometimes make the structure worse). At this point I would go through the lists of outliers (i.e. residues with bad statistics) from validation and try to fix them individually. (You're probably not going to be able to correct every single problem - don't worry, it can be very difficult to get a "perfect" structure at this resolution.) Use Coot's Ramachandran restraints in real-space refinement, but be sure to inspect the conformation afterwards to see if it makes sense. Make sure you run the real-space refinement every time you adjust the model; this will make it much easier for phenix.refine the next time you run it. Without knowing more about your structure I don't know if you need to change your phenix.refine settings, but default settings plus weight optimization is a good start. *Do not* use the Ramachandran restraints in Phenix at this point. Also, as Pavel suggested, you may benefit from updating to the latest nightly build. -Nat

On Thu, Aug 1, 2013 at 7:59 AM, miyatake wrote:

Are there GUI tabs for DEN refinement in the current version of PHENIX?

Yes, but it's still flagged as experimental pending further debugging of the underlying program. To enable it, open the preferences, check the box labeled "Enable alpha-test programs and features", and restart Phenix. The DEN refinement GUI should now appear below the regular refinement button. -Nat