Hi John, are these waters agree with the density? All phenix.refine is using to decide about whether to keep or remove a water is: - density (mFo-DFc and 2mFo-DFc) peak height and shape; - distance between the density peak and surrounding atoms. The corresponding control parameters are exposed to users, so one can make them softer or stricter. If phenix.refine deletes a water that only means that one or a few selection criteria are not fulfilled. You can relax water filtering criteria at the risk of adding more spurious waters. Or you let phenix.refine do what it does, but at the very end add missing waters and do a round of refinement without water update so your waters are kept. I'm actually puzzled about this a bit. If you send me the PDB files (before and after phenix.refine run, and tell which waters are missing) then I will have a closer look. I also need the data file (so I can run refinement myself). Please send files off-list, to my email. Pavel On 4/3/12 8:39 AM, John Pascal wrote:

Hi Pavel.

The waters around the metals agree quite well with previously determined structures, and some of the waters are included in the metal.edits file describing the expected metal-water distances. These same waters are sometimes removed during the routine, and their residue number is given to a different water residue that has been selected at a different location in the structure. So in subsequent rounds using the same metal.edits file introduces a model distance that is no where near the distance defined in the .edits file, and causes program termination. I can of course remedy this, but my feeling is that the metal sites should be handled carefully by me, and then left alone as the rest of the structure is tested for additional waters. Thanks.

Best, John

On 4/3/12 11:12 AM, Pavel Afonine wrote:

...

Hi John,

no, not at the moment. Only turning water update off will leave the water alone.

Could you please explain why you would like to do this?

Pavel

On 4/3/12 8:01 AM, John Pascal wrote:

...

Hello All,

While using Update Waters in the phenix GUI, several waters that have been defined around a metal binding site are removed. I am happy with the placement of these waters - can I specify somewhere that Update Waters should leave them alone? and that their positions, etc. are still refined during the run? Thanks.

Best Regards, John

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What would be really great is to improve the algorithm so it does a better job, and for that I need a real example illustrating what exactly is not right. Providing functionality to keep waters may be good to have, but on the other hand it's going to be another handle for performing manual job, and phenix.refine has already too many handles, which is fine, but automation is the goal. Pavel On 4/3/12 9:15 AM, Nathaniel Echols wrote:

...

are these waters agree with the density? All phenix.refine is using to decide about whether to keep or remove a water is: - density (mFo-DFc and 2mFo-DFc) peak height and shape; - distance between the density peak and surrounding atoms. The corresponding control parameters are exposed to users, so one can make them softer or stricter. If phenix.refine deletes a water that only means that one or a few selection criteria are not fulfilled. It is often not obvious which of these criteria need to be changed. Using the default settings, I always see additional peaks left over

On Tue, Apr 3, 2012 at 8:50 AM, Pavel Afonine wrote: that to my eye look like waters, and it almost always removes waters that I am convinced were placed correctly. I agree with John - it would be nice to be able to specify that the routine should only add waters and not remove any that were in the input file. At the very least, it should not be removing waters that have bond restraints.

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On Tue, Apr 3, 2012 at 9:40 AM, Engin Özkan wrote:

that now. Moreover, it used to be that links/restraints from previous GUI runs would be silently kept by default, and in your next phenix.refine run, your restraints are just plain wrong (I think Nat has changed or is changing that behavior, thanks!).

If you generate new restraints in the current nightly builds, it will handle them more sensibly. Restoring parameters from runs in older versions will still keep the previously defined restraints, but there are now multiple ways to edit and/or delete them - previously it was much less obvious. -Nat

Hi Mark,

I need some help advice regarding anisotropic refinement, which I've never done before. My current Rfactors are R-work = 0.1587, R-free = 0.1726, resolution 1.26 Å.

two things I would do right away without thinking long: - add H atoms; - refine all macromolecular atoms with anisotropic ADPs (probably, except alternative conformations), and solvent with isotropic ADPs; Also, please make sure to use recent Phenix version. That may have impact in your case, since latest phenix.refine have improved handling of hydrogen atoms (*), and a new bulk-solvent and anisotropic scaling procedure (**). If you still want to define by hand which atoms to refine isotropically/anisotropically, then a parameter file like this should allow to do this: refinement { refine { adp { individual { isotropic = (chain A and resseq 1:100) or (chain X and resseq 300:900) or water anisotropic = (chain Z and resseq 123 and name CG1) or (element Au) } } } } Make sure you cover all atoms with isotropic = ... anisotropic = ... selections, because the default behavior is that if you change these parameters then phenix.refine assumes you know what you do. If you do not select all atoms with these selections, then B-factors of atoms that are not selected will not be refined. I don't like this and I will change this once I get a chance. For your specific example: refinement { refine { adp { individual { isotropic = not (chain A and (resseq 7:44 or resseq 61:149 or resseq 173:182 or resseq 202:254 or resseq 263:290)) anisotropic = chain A and (resseq 7:44 or resseq 61:149 or resseq 173:182 or resseq 202:254 or resseq 263:290) } } } } I've never tried but this might work too: refinement { refine { adp { individual { aniso_sel = chain A and (resseq 7:44 or resseq 61:149 or resseq 173:182 or resseq 202:254 or resseq 263:290) isotropic = not $aniso_sel anisotropic = $aniso_sel } } } } Phil experts may comment on this more. Let me know if you have any questions or need any help with this! Good luck, Pavel (*) "On contribution of hydrogen atoms to X-ray scattering" http://www.phenix-online.org/newsletter/ (**) http://www.phenix-online.org/presentations/PhenixSantaFe2012_PA.pdf

I have TLS groups, and know I need to exclude these from individual anisotropic refinement.

Questions:

1) how to decide my atom selection for anisotropic refinement. I've identified segments with MC B below 20, but what to do for surface residues which have well ordered MC but B factors for side chains increase (e.g. along a well ordered Lys side chain) - should (can?) I

a) refine only MC anisotropically, b) refine whole residue anisotropically, c) refine isotropic

2) How to write a a parameter file for phenix.refine defining the atom selections.

The residues I'd like to use for anisotropic refinement are: A7-A44 A61-A149 A173-A182 A202-A254 A263-A290 all in chain A

I found following on phenix.refine online docs, but am not sure to to extend this for a more complex selection needed above, which Ideally I'd put into a file to be read in when I start refinement. Could yo show me how to do for A7-A44 and A61-A149, and I'll follow example for the rest.

adp.individual.anisotropic="resid 1:2 and not element H" adp.individual.isotropic="not (resid 1:2 and not element H)

Thanks for help.

Mark .