[phenixbb] Low resolution refinement

Pavel Afonine pafonine at lbl.gov
Sat Apr 26 09:31:47 PDT 2014

A few more points:

- define secondary structure and use it in refinement. If you don't use 
secondary structure restraints then even ideal secondary structure in 
starting model will deteriorate after refinement at such low resolution. 
Most importantly, make sure that secondary structure restraints that you 
defined are actually used in refinement. If you suspect they are not 
used in refinement - please report a bug right away.
You can use phenix.secondary_structure_restraints to let Phenix 
automatically identify secondary structure. It is critical that you 
manually review it, and edit if necessary (which most likely will be the 
case) in order to get the most accurate annotation!

- while torsion NCS is a better option most of the time, at very low 
resolution Cartesian NCS might be a betetr option. So, as Nat pointed 
out, try both and see which one works best.

- if real-space refinement does not perform well in your case, please 
report a bug, otherwise use it as it will eliminate rotamer outliers. 
Again, if there is any suspicion that it does not perform as expected - 
please report a bug.

- if there are Ramachandran plot outliers: fix them all manually fist, 
then use Ramachandran plot restraints to prevent them from re-occurring 
(Important: do not use Ramachandran plot restraints to fix the outliers!).

- if data set is severely incomplete - look at two 2mFo-DFc map: regular 
and missing Fobs "filled" (phenix.refine outputs both maps).

Good luck!

On 4/26/14, 7:59 AM, Nathaniel Echols wrote:
> On Fri, Apr 25, 2014 at 3:15 PM, Appu kumar <appu.kumar9 at gmail.com 
> <mailto:appu.kumar9 at gmail.com>> wrote:
>                      I want to refine the low resolution structure
>     (6.5A). It is a membrane protein and we are able to phase it. It
>     will be great help if any one can guide me how to proceed further
>     with the refinement. People uses Phenix to refine low resolution
>     structure, so phenix is really a good program to proceed for
>     refinement against low resolution.
> How to proceed depends on what exactly you want the refinement to do. 
>  If the structure is already approximately correct with no major 
> conformational changes, here are some basic guidelines:
> 1) Use tight restraints.  The weight optimization doesn't work very 
> well in this resolution range (at least in my hands), so setting very 
> low values for wxc_scale and wxu_scale (which control the weighting of 
> the X-ray term relative to the geometry and B-factor restraints, 
> respectively) is a good place to start.  I would try values of 
> wxc_scale=0.025 and wxu_scale=0.05, but you may need to experiment 
> with this.
> 2) Use NCS restraints if you have multiple copies in the ASU.  (The 
> torsion restraints are usually sufficient - and much easier to use - 
> but it might be worth trying cartesian restraints too.)
> 3) Use a reference model if you have a good high-resolution structure 
> available (this can be just part of the structure).
> 4) Real-space refinement is probably not going to work.
> 5) Try using a very conservative refinement strategy at first - for 
> instance, rigid-body and group B-factor refinement.  The latter should 
> work better now (in the latest nightly builds, and imminent 1.9 
> release) since Pavel added restraints on neighboring groups.  You can 
> also try refining TLS for domains.  My own experience was that 
> individual coordinate and B-factor refinement worked best - the latter 
> is somewhat controversial but with tight restraints it ends up not 
> being very different from refining an overall B-factor.  I also found 
> it helpful to reset all B-factors to 20 at the start of refinement to 
> remove bias from the starting model.
> 6) Be very paranoid about the 2mFo-DFc map - at this resolution it 
> will look almost exactly like your model.  Make lots of omit maps, and 
> if you have any heavy scatterers (metals, etc.) those will be very 
> helpful in confirming that you have the correct structure.
> If you actually have to do a lot of rebuilding, it is considerably 
> more difficult; DEN or Rosetta refinement may be helpful.
> -Nat

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