[phenixbb] Re: [EXTERNAL] Re: Refine pixel size for EM map?

Hi Reza, It was tested on large assemblies (the vault particle, which is 70nm). It is not something I would do with every refinement, but for calibrating the pixel size of a particular scope at a given mag. Generally it should be stable at least for months unless the scope has some major work done. It is the same procedure (in effect) as we would previously do manually in Chimera, measuring the real space correlation of a crystal structure at different nominal pixel sizes, but just performed automatically and a lot faster. Cheers Oli

On Aug 1, 2025, at 6:17 PM, Reza Khayat wrote:

Hi,

Very cool; however, I'm curious if the procedure was tested on large assemblies. I'd imagine that a small error in pixel (0.001Angstrom) would propagate into a significant error (1Angstrom) at the periphery for large assemblies (~100nm). Would, a 1Angstrom change may be interpreted as biologically relevant? Also, is it recommended that every refinement include this process, or should the refinement be restricted to a magnification of a specific microscope? Perhaps its best for me to wait for the publication.

Best wishes, Reza From: Oliver Clarke Sent: 01 August 2025 1:57 PM To: Pavel Afonine Cc: PHENIX user mailing list Subject: [EXTERNAL] [phenixbb] Re: Refine pixel size for EM map?

Huge thanks to Pavel & Randy for their patience and for developing such useful tools! We will use phenix.magref a lot! Happy friday all!

Cheers Oli

...

On Aug 1, 2025, at 1:51 PM, Pavel Afonine wrote:

Happy Friday, Phenix'ers!

Many thanks to Oli Clarke, who finally encouraged us to made a pixel size calibration tool in Phenix accessible and who offered an incredible amount of help by critically testing it and helping to weed out bugs!

Here we go. The following command:

phenix.magref model.pdb map.mrc resolution=3.3

will take your map (map.mrc) and a reference atomic model (model.pdb), and calibrate the pixel size (magnification) by sampling plausible calibration scales. For each trial value, it performs extensive rigid body refinement. As a result, it will output the corrected map for you to use from this point on, as well as the best-fitting atomic model with corrected box information (CRYST1, if you are still in the PDB format world).

It is rather fast too: for something as large as 240k atoms (30k residues) and 800x800x800 map it takes about 6.5 minutes and for anything smaller times usually range from tens of seconds to a minutes or a few minutes.

IMPORTANT: The reference model used for pixel size refinement should not have been refined or flexibly fitted to a cryo-EM map. It is expected to be a closely matching model determined by crystallography and should be approximately placed (docked) into the map.

This is available in latest nightly builds of Phenix (e.g., 2.0-5774 and up). Let me know should you have any questions or encounter any problems!

Pavel

P.S. Another way to achieve this in Phenix is to use a similar tool from Phaser:

phenix.python <path to phenix>/modules/phaser_voyager/src/New_Voyager/scripts/emplace_rigid_body.py --map map.mrc --model_file model.pdb --d_min 3 --refine_scale

On 6/3/25 00:19, Oliver Clarke wrote:

...

Hi,

I remember asking about this some time ago, but I am not sure if it was ever implemented.

Is there a way to refine just pixel size for an EM map in phenix.real_space refine (or another phenix utility)? Even just automatically rigid-body fitting a crystal structure and calculating the pixel size with max correlation would be handy. We do this at the moment using Chimera/ChimeraX, but an automated way to do it in phenix would be handy.

In the absence of a crystal structure, I wonder if directly refining pixel size during real space refinement would be possible/reasonable at higher resolution (<2Å)?

Cheers Oli _______________________________________________ phenixbb mailing list -- [email protected] To unsubscribe send an email to [email protected] Unsubscribe: phenixbb-leave@%(host_name)s

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