Hi Pavel, I have 2 important waters at 2.12 A distance after refinement. They are ligands of Mg2+. How can I restrain this distance to 2.3A (to pass pdb validation)? Is it in the ordered solvent that I need to change the default h_bond_min_mac = 1.8 h_bond_min_sol = 1.8 h_bond_max = 3.2 Maia Pavel Afonine wrote:

Hi,

phenix.refine removes F-obs outliers for refinement. See .log file for the number of such reflections. Also, it automatically removes Fobs=0. Do phenix.mtz.dump data.mtz to see the content of your MTZ file.

Pavel.

On 3/27/10 1:26 PM, r n wrote:

...

Hi all I observed that for the same data set and resolution, number of reflections for the refinement is different values both in phenix (9488), refmac5 (9423) and refmac using coot interface (9530).

Please anyone can let me know why the differences between the programs for same resolution and data.

Thanks ram

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Hi Pavel, I have done the things that you suggested, and I have all the restraints for the Mg2+ and I see those 2 waters, but they are at 2.12A distance. I think there should be some general restraints that should keep h_bonds at least at > 2.2A. That is why I asked you about those defaults (1.8A etc). Why are they at 1.8A? Should not they be at least at 2.2A? Maia Pavel Afonine wrote:

Hi Maia,

I assume that these waters are already present in your structure.

Then run phenix.phenix.metal_coordination model.pdb

That will create a restraints file for you Mg2+. Edit it, if necessary, to satisfy your expectations. These two waters should be there.

Run phenix.refine and give it that restraints file. If you run phenix.refine with "ordered_solvent=true" option, then phenix.refine may or may not remove these two waters. If you absolutely need to keep these waters, run phenix.refine with "ordered_solvent=true" untill the last moment, then add two waters and run it again with "ordered_solvent=false". Something like this...

Pavel.

On 3/27/10 3:50 PM, Maia Cherney wrote:

...

Hi Pavel,

I have 2 important waters at 2.12 A distance after refinement. They are ligands of Mg2+. How can I restrain this distance to 2.3A (to pass pdb validation)? Is it in the ordered solvent that I need to change the default h_bond_min_mac = 1.8 h_bond_min_sol = 1.8 h_bond_max = 3.2

Maia

Pavel Afonine wrote:

...

Hi,

phenix.refine removes F-obs outliers for refinement. See .log file for the number of such reflections. Also, it automatically removes Fobs=0. Do phenix.mtz.dump data.mtz to see the content of your MTZ file.

Pavel.

On 3/27/10 1:26 PM, r n wrote:

...

Hi all I observed that for the same data set and resolution, number of reflections for the refinement is different values both in phenix (9488), refmac5 (9423) and refmac using coot interface (9530).

Please anyone can let me know why the differences between the programs for same resolution and data.

Thanks ram

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Hi Pavel, what is h_bond_min_mac and h_bond_min_sol. which one should I change to 2.2 so that it will not affect the coordination distances of Mg2+ (around 2A)? Maia Pavel Afonine wrote:

Hi Maia,

...

I have done the things that you suggested,

great!

...

and I have all the restraints for the Mg2+ and I see those 2 waters, but they are at 2.12A distance.

Are these two waters located in the centers of corresponding density peaks? If this is the case then I don't see the subject of your worries. If this is not the case (these waters are off the peaks) then edit the file that defines restraints for water-Mg2+ interactions.

...

I think there should be some general restraints that should keep h_bonds at least at > 2.2A.

Yes. You wrote it yourself in your previous email:

h_bond_min_mac = 1.8 h_bond_min_sol = 1.8 h_bond_max = 3.2

Just change the values to what you like!

...

That is why I asked you about those defaults (1.8A etc). Why are they at 1.8A? Should not they be at least at 2.2A?

Honestly, I don't remember why I put these values (this is pretty old code in phenix.refine that I wrote several years ago). All I recall is that I had a reason for this. I probably need to run through the whole PDB and see how the density peaks (actual, residual, omit-residual) are distributed w.r.t. macromolecule, and write it somewhere for the future reference. This could be a project for a few weeks, but it will definitely yield a rock-solid conclusion about "good" distances. Meanwhile feel free to edit the available parameters.

By the way, this is what CNS is using:

{* minimum distance between water and any atom *} {===>} min=2.6; {* maximum distance between water and any atom *} {===>} max=4.0;

{* minimum hydrogen bonding distance between water and O or N *} {===>} hmin=2.0; {* maximum hydrogen bonding distance between water and O or N *} {===>} hmax=3.2;

Pavel.

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Hi Pavel, I have waters that are part of the Mg2+ coordination. They should be at ~2.1A from Mg2+. At the same time, they should be at a hydrogen-bonding distance from each other (minimum 2.2A otherwise the validation at the PDB complains). The two waters after refinement without restraints had closer (2.12A) distance between them, so I managed to restrain the distance to 2.23A using pdb_interpretation. But my question was, should not the refinement automatically restrain close distances between any atoms, including waters? (By the way, I picked them manually). Maia Pavel Afonine wrote:

Hi Maia,

I'm not sure I understand the question. If you have that specific water in your structure already (and you run phenix.refine with "ordered_solvent=false"), then changing the h_bond_min_mac or h_bond_min_sol will not change anything since these parameters are used in water update.

However, if you are at the stage of water picking and the distance between Mg2+ and prospective water peak is 2.2A then the water will be placed there (providing that the corresponding peak satisfies the other criteria).

Could you please explain what exactly the problem is, and then I will try my best to give you the solution?

Thanks! Pavel.

On 3/27/10 5:23 PM, Maia Cherney wrote:

...

Hi Pavel, what is h_bond_min_mac and h_bond_min_sol.

which one should I change to 2.2 so that it will not affect the coordination distances of Mg2+ (around 2A)?

Maia

Pavel Afonine wrote:

...

Hi Maia,

...

I have done the things that you suggested,

great!

...

and I have all the restraints for the Mg2+ and I see those 2 waters, but they are at 2.12A distance.

Are these two waters located in the centers of corresponding density peaks? If this is the case then I don't see the subject of your worries. If this is not the case (these waters are off the peaks) then edit the file that defines restraints for water-Mg2+ interactions.

...

I think there should be some general restraints that should keep h_bonds at least at > 2.2A.

Yes. You wrote it yourself in your previous email:

h_bond_min_mac = 1.8 h_bond_min_sol = 1.8 h_bond_max = 3.2

Just change the values to what you like!

...

That is why I asked you about those defaults (1.8A etc). Why are they at 1.8A? Should not they be at least at 2.2A?

Honestly, I don't remember why I put these values (this is pretty old code in phenix.refine that I wrote several years ago). All I recall is that I had a reason for this. I probably need to run through the whole PDB and see how the density peaks (actual, residual, omit-residual) are distributed w.r.t. macromolecule, and write it somewhere for the future reference. This could be a project for a few weeks, but it will definitely yield a rock-solid conclusion about "good" distances. Meanwhile feel free to edit the available parameters.

By the way, this is what CNS is using:

{* minimum distance between water and any atom *} {===>} min=2.6; {* maximum distance between water and any atom *} {===>} max=4.0;

{* minimum hydrogen bonding distance between water and O or N *} {===>} hmin=2.0; {* maximum hydrogen bonding distance between water and O or N *} {===>} hmax=3.2;

Pavel.

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Hi Maia, not sure if it makes sense to me, but anyway, technically you can do that: just define a custom bond between these two atoms as described in: http://www.phenix-online.org/version_docs/1.6.1-353/refinement.htm#anch80 And to make sure phenix.refine doesn't remove any of these two waters, run it with "ordered_solvent=true". Pavel. On 3/27/10 4:55 PM, Maia Cherney wrote:

Pavel, I just want to restrain the distance between 2 particular waters. Is it in the pdb_interpretation? Maia