Dear Ina Have a look here (from the CCP4BB) http://www.mail-archive.com/[email protected]/msg19631.html Best regards, Folmer Fredslund 2011/2/17 Ina Lindemann :

Hi,

I am refining a crystal structure of a protein with 198 amino acids at 1.59 A resolution. I have used the function Update waters in phenix. After visual inspection of the found water molecules I deleted some of them, but there are still 334 water molecules left which show reasonable electron density. Many of them interacts just with other water molecules and not with the protein. I calculated the average B of water molecules = 26.3 and protein = 12.6. Now I am wondering if the number of water molecules and their average B value is to high with respect to the protein and would be very pleased about your answers.

With best regards, Ina

Ina Lindemann Philipps-Universität Marburg Pharmazeutische Chemie AG Klebe Marbacher Weg 6 35032 Marburg Tel.: 06421/2825908

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Hi Ina, I'm not sure why you think it is unrealistic.. The solvent content in macromolecular crystals may reach 80% or so. Yes, most of it is disordered, but the line is pretty subtle - I'm not sure you can reliably say how many waters you will find. Not to forget - the amount of water you will fins also depends on data and current model quality. Just to mention: phenix.refine has fairly thorough protocol for updating the ordered solvent model during refinement process. Updating the ordered solvent model involves the following steps: 1) Elimination of waters present in the initial model based on user-defined cutoff criteria on ADP, occupancy and inter-atomic distances (water-water, macromolecule-water), (2mFobs-DFmodel) density values at water oxygen centers, map correlation coefficient values computed for each water oxygen atom. 2) Location of new peaks in (mFobs-DFmodel) map, with following filtering of these peaks by their height and distance to other atoms. The remaining peaks after filtering are considered as new water oxygens. It is possible to specify which type of ADP for newly added waters will be refined: isotropic or anisotropic. 3) Depending on the refinement strategy (typically at high resolution), individual isotropic or anisotropic B-factors of newly added water molecules are refined prior the refinement of all other parameters. This is important to do since the newly placed waters have approximate values of B-factor (which is usually the average B) and if a large number of new waters is added at once this may significantly increase the R-factors at this step and slow down the refinement convergence. The higher resolution, the stronger the effect. 4) Unlike macromolecular atoms that are ‘wired’ to each other through the geometry restraints, the electron density is the only force to keep the already added water in place, and occasionally it may happen that the density peak is not strong enough to keep water in it and the water may drift away from the peak during refinement. Specific algorithm implemented in phenix.refine that prevents this from happening. Pavel. On 2/16/11 11:52 PM, Ina Lindemann wrote:

Hi,

I am refining a crystal structure of a protein with 198 amino acids at 1.59 A resolution. I have used the function Update waters in phenix. After visual inspection of the found water molecules I deleted some of them, but there are still 334 water molecules left which show reasonable electron density. Many of them interacts just with other water molecules and not with the protein. I calculated the average B of water molecules = 26.3 and protein = 12.6. Now I am wondering if the number of water molecules and their average B value is to high with respect to the protein and would be very pleased about your answers.

With best regards, Ina