Dear Pavel, Thank you for your prompt response. We will try and report back on results. Just a small clarifying question -

reference_coordinate_restraints.enabled=true

reference_coordinate_restraints.selection=water

reference_coordinate_restraints.sigma=0.2

I shall look and set in Real-space refinement protocol? Many thanks. Sincerely, Dmitry On January 20, 2024 5:25:24 AM Pavel Afonine wrote:

Hi Dmitry,

Here are the considerations regarding water picking in cryo-EM maps:

Unlike crystallography, where changing the model changes the maps (e.g., adding more structure improves the maps), in cryo-EM, changing the model does not change the map. Therefore, tasks such as refining B factors and adding water are best done using the most complete and accurate atomic model of the macromolecule, preferably towards the end.

Once you have added water, you need to run a round of refinement and check all water manually. While automated tools generally add water reasonably well, there is no 100% guarantee that all water will be correct. Typically, you perform this step once towards the end.

"Flying water" seems to be an issue indeed. While I'm working on the solution, a quick and easy workaround for you to use now is to restrain water positions to their original coordinates using weak harmonic restraints. Keywords you'll need:

reference_coordinate_restraints.enabled=true reference_coordinate_restraints.selection=water reference_coordinate_restraints.sigma=0.2

(also can be set up in the GUI).

Let me know if you have any questions or need help!

Good luck! Pavel

On 1/19/24 03:33, Dmitry Semchonok wrote:

...

Dear colleagues,

Please share your experience with how you handle the water molecules.

So, we have a refined model and added the water molecules using the Douse protocol. So, should we refine the model again afterwards? Or is the model considered ready?

We tried to refine our model with water, but then the water molecules flew away from the places.

Please let me know what is the correct way to proceed.

Many thanks for considering my request.

Sincerely,

Dmitry

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