Hi Pavel, Thanks a lot as usual. I learnt that one can also make Refmac output like that, but it is not the default. So, that confusion is clear. At your leasure, if you could explain a bit about the water picking parameters, that would be great. In my case, for 132 residues and 55% solvent content, the default setting was picking 330 waters, which is bit too much I felt. Did change the b_iso_min = 10 and b_iso_max = 40, just to see what happens.. (because I thought for ordered water the later should not be too high..) but I was not sure what the other parameters mean. Like pick water above 3 sigma, remove below 0.9 sigma as we are used to from Arp.. No comparison between the different programs and what their keywords are.. just a feeling that if some of these key words are explained at the last section instead of True/False in Red / Blue, it could help the users, just as b_iso_min/max is explained.. Best, Partha On 10/9/07, Pavel Afonine wrote:

Hi Partha,

...

I am bit confused with two things. First I did specify individual isotopic, as following, still see ANAISOU in PDB. Is it from TLS strategy? I have used TLS in refmac, don't see it there.

Any atom participating in a TLS group will have ANISOU record. This is explained in the Manual: http://phenix-online.org/documentation/refinement.htm paragraph "Refinement of atomic displacement parameters (commonly named as ADP or B-factors)".

You don't see this in Refmac because Refmac does not output this right (= as efficient as it should be).

...

Second thing is, I want to tighten the water picking. How can I do that?

1) http://phenix-online.org/documentation/refinement.htm paragraph "Water picking".

2) Below is the extract with default water picking parameters related to your question:

refinement { ordered_solvent { b_iso_min = 1 b_iso_max = 50 primary_map_cutoff = 3 secondary_map_cutoff = 1 min_solv_macromol_dist = 1.8 max_solv_macromol_dist = 6 min_solv_solv_dist = 1.8 } }

To tighten the water picking I would change it like this (for example; depending on model quality, resolution):

refinement { ordered_solvent { b_iso_min = 10 b_iso_max = 50 primary_map_cutoff = 3.5 secondary_map_cutoff = 1.5 min_solv_macromol_dist = 1.8 max_solv_macromol_dist = 4 min_solv_solv_dist = 1.8 } }

Try several values and see the effect -- you will get less waters and more confidence that they are all real waters. I can explain some more about each parameter, if necessary.

As usual, let us know if you have any questions.

Pavel.

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Cheers Pavel, 160 water molecules from 330.. P On 10/9/07, Pavel Afonine wrote:

Hi Partha,

...

At your leasure, if you could explain a bit about the water picking parameters, that would be great. In my case, for 132 residues and 55% solvent content, the default setting was picking 330 waters, which is bit too much I felt. Did change the b_iso_min = 10 and b_iso_max = 40, just to see what happens.. (because I thought for ordered water the later should not be too high..) but I was not sure what the other parameters mean. Like pick water above 3 sigma, remove below 0.9 sigma as we are used to from Arp..

refinement { ordered_solvent { b_iso_min = 1 b_iso_max = 50 primary_map_cutoff = 3 secondary_map_cutoff = 1 min_solv_macromol_dist = 1.8 max_solv_macromol_dist = 6 min_solv_solv_dist = 1.8 } }

Here are the "most important":

1) Waters with refined B-factors beyond this interval are removed: (b_iso_min = 1, b_iso_max = 50). I presume you do not refine the occupancies for water molecules; otherwise there are similar parameters for occupancies as well.

2) phenix.refine uses two maps to select prospective electron density peaks: mFo-DFc (primary map) and 2mFo-DFc (secondary map). primary_map_cutoff = 3 and secondary_map_cutoff = 1 define peak heights; default is -- a peak must be higher than 3 sigma for primary map and higher than 1 sigma for secondary map to be considered as a water pick-candidate. Raising these values will select stronger peaks and obviously will result in lesser number of found waters.

3) Only peak located within this distance interval from macromolecule are considered (min_solv_macromol_dist = 1.8, max_solv_macromol_dist = 6). Some people believe that 6.0A is too generous. You may try to reduce it to lesser values like 3.5...4.5A. Again this will reduce the number of found waters.

4) Waters separated by less than min_solv_solv_dist = 1.8 are removed.

The options "2)" and "3)" can have the highest impact on the number of located water molecules. So, I would play with those first and monitor R/Rfree as well.

The other parameters have higher "expert level" and lesser impact on the outcome, so I don't explain them until requested.

Pavel.

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