This is the output when I use the `sequence_file` keyword: ``` data_manager { sequence_files = "2xoa.fasta" default_sequence = "2xoa.fasta" real_map_files = "cryosparc_P4_J203_map.mrc" real_map_files = "cryosparc_P4_J272_map.mrc" real_map_files = "cryosparc_P4_J270_map.mrc" default_real_map = "cryosparc_P4_J203_map.mrc" model { file = "2xoa_fit_j203.pdb" } default_model = "2xoa_fit_j203.pdb" } em_placement { map_model { full_map = cryosparc_P4_J203_map.mrc half_map = cryosparc_P4_J270_map.mrc half_map = cryosparc_P4_J272_map.mrc } docking { refine_cell_scale = true } biological_unit { molecule { model_file = 2xoa_fit_j203.pdb } } } Starting job =============================================================================== Sorry: Please supply a sequence file ```

On Jun 4, 2025, at 12:48 PM, Oliver Clarke wrote:

Hi Randy,

Thanks!! Voyager.em_placement looks like it should do the trick - however, when I run it, it complains it needs a sequence file. When I provide a seqence file, e.g.:

phenix.voyager.em_placement model_file=2xoa_fit_j203.pdb full_map=cryosparc_P4_J203_map.mrc refine_cell_scale=true half_map=cryosparc_P4_J270_map.mrc half_map=cryosparc_P4_J272_map.mrc sequence_files=2xoa.fasta

... it seems to read the sequence file per the input parameters, but then still complains it needs a sequence file. Per Tom's message yesterday regarding map_sharpening, I tried the seq_file flag instead, but that does not seem to exist for this utility. Is there another sequence keyword I am missing?

Cheers Oli

...

On Jun 4, 2025, at 11:55 AM, Randy John Read wrote:

Hi Oli,

I think this has come up before, but as far as I know the only current method to refine pixel size in Phenix is by using the refine_cell_scale option in phenix.voyager.em_placement. This requires you to have a placed (or at least dockable) model that is on the right scale (i.e. hasn’t been refined against the map of interest). These days, the high-confidence subset of an AlphaFold model would probably be sufficient to give a good correction for the magnification factor. In tests I’ve done, it has a pretty high convergence radius.

What you probably want is something that will correct a model that may already have been refined against the map of interest. This is a feature that Pavel Afonine and I have discussed before, and I think Pavel did at least some preliminary experiments in phenix.real_space_refine. Like you, I think it should be possible to use the geometric restraints to get the refinement engine to correct the pixel size to make it compatible with good geometry. However, this may not be trivial to get to work robustly and might require things like taking account of second derivative terms relating the positional and pixel size (or magnification factor) parameters.

Pavel might have more to say on this.

Best wishes,

Randy

...

On 3 Jun 2025, at 08:19, 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

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

Sorry, should have posted a script before looking away from the BB! I’m glad you got there. We should probably add it as an advanced option to the GUI for the program, including access to the rigid-body refinement option as an alternative to a full docking search. The way it works, it just adjusts the rigid-body positioning of the model while refining the scale factor relating the map to the model. The increase in LLG during this refinement will tell you something indirectly about the significance of the change, but not the precision. If this is a tool people will want to use (considering that calibration of pixel size is getting much better these days during the experiment), I could implement something where, after the optimum has been found, several refinements fixing various perturbed pixel sizes could be done to see how the LLG varies, which would give a plot to look at. Best wishes, Randy

On 4 Jun 2025, at 11:58, Oliver Clarke wrote:

Nevermind, got it, there is another keyword:

phenix.voyager.em_placement model_file=2xoa_fit_j203.pdb full_map=cryosparc_P4_J203_map.mrc refine_cell_scale=true half_map=cryosparc_P4_J270_map.mrc half_map=cryosparc_P4_J272_map.mrc map_model.sequence_composition=2xoa.fasta biological_unit.molecule.molecule_name=2xoa

And then the scale value is in the log, and matches very well with what we estimate using Chimera. Is there a plot generated somewhere of the scale vs correlation, TFZ etc? I couldn't find such in the log or output directory.

Cheers Oli

...

On Jun 4, 2025, at 12:50 PM, Oliver Clarke wrote:

This is the output when I use the `sequence_file` keyword:

``` data_manager { sequence_files = "2xoa.fasta" default_sequence = "2xoa.fasta" real_map_files = "cryosparc_P4_J203_map.mrc" real_map_files = "cryosparc_P4_J272_map.mrc" real_map_files = "cryosparc_P4_J270_map.mrc" default_real_map = "cryosparc_P4_J203_map.mrc" model { file = "2xoa_fit_j203.pdb" } default_model = "2xoa_fit_j203.pdb" } em_placement { map_model { full_map = cryosparc_P4_J203_map.mrc half_map = cryosparc_P4_J270_map.mrc half_map = cryosparc_P4_J272_map.mrc } docking { refine_cell_scale = true } biological_unit { molecule { model_file = 2xoa_fit_j203.pdb } } }

Starting job =============================================================================== Sorry: Please supply a sequence file ```

...

On Jun 4, 2025, at 12:48 PM, Oliver Clarke wrote:

Hi Randy,

Thanks!! Voyager.em_placement looks like it should do the trick - however, when I run it, it complains it needs a sequence file. When I provide a seqence file, e.g.:

phenix.voyager.em_placement model_file=2xoa_fit_j203.pdb full_map=cryosparc_P4_J203_map.mrc refine_cell_scale=true half_map=cryosparc_P4_J270_map.mrc half_map=cryosparc_P4_J272_map.mrc sequence_files=2xoa.fasta

... it seems to read the sequence file per the input parameters, but then still complains it needs a sequence file. Per Tom's message yesterday regarding map_sharpening, I tried the seq_file flag instead, but that does not seem to exist for this utility. Is there another sequence keyword I am missing?

Cheers Oli

...

On Jun 4, 2025, at 11:55 AM, Randy John Read wrote:

Hi Oli,

I think this has come up before, but as far as I know the only current method to refine pixel size in Phenix is by using the refine_cell_scale option in phenix.voyager.em_placement. This requires you to have a placed (or at least dockable) model that is on the right scale (i.e. hasn’t been refined against the map of interest). These days, the high-confidence subset of an AlphaFold model would probably be sufficient to give a good correction for the magnification factor. In tests I’ve done, it has a pretty high convergence radius.

What you probably want is something that will correct a model that may already have been refined against the map of interest. This is a feature that Pavel Afonine and I have discussed before, and I think Pavel did at least some preliminary experiments in phenix.real_space_refine. Like you, I think it should be possible to use the geometric restraints to get the refinement engine to correct the pixel size to make it compatible with good geometry. However, this may not be trivial to get to work robustly and might require things like taking account of second derivative terms relating the positional and pixel size (or magnification factor) parameters.

Pavel might have more to say on this.

Best wishes,

Randy

...

On 3 Jun 2025, at 08:19, 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

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk