Hi Louis, Greg,

There are two main reasons why I still recommend some post-refinement of models from ISOLDE before deposition:

1. ISOLDE currently doesn't attempt any B-factor refinement at all, so without some post-refinement the B-factors in your output model will be whatever they were in the model(s) you used as your starting point.
2. The AMBER force field used by ISOLDE is great in many ways, but it doesn't quite replicate the fine details of equilibrium bond lengths and angles seen in ultra-high resolution structures - due to various systematic differences your model will always be flagged for high RMSDs in these metrics.

All that being said, yes: I've often observed the same behaviour as you. That's why I included some options (under the "isolde write" command) to write out input scripts designed to limit the refinement as much as possible to only the above (tightening bond lengths/angles and refining B-factors, with all more "aggressive" options disabled). See for example https://tristanic.github.io/isolde/static/isolde/doc/commands/isolde.html#isolde-write-phenixrsrinput for the cryo-EM case (to get the equivalent documentation within ChimeraX, do "usage isolde write" then click the applicable link in the log).

Best,
Tristan


On Thu, 4 Dec 2025 at 22:57, Greg Pintilie via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:

I have noticed the same thing for such high resolution maps… so I lately I have been depositing ISOLDE refined models. I don’t think there is a strict reason to deposit models refined with one method or another.

As to the reason, it could be that Phenix uses stronger restraints for backbone dihedrals (i.e. may have lower Ramachandran outliers) and rotamers (less unfavored rotamers). But this may make side chains not fit the density as well. On the other hand, ISOLDE models seem to fit the density better and have lower clash scores. If you want to stick with Phenix there are probably some parameters you can tweak to make these restraints not as strong perhaps?

Greg



> On Dec 4, 2025, at 3:02 PM, man6ny--- via ChimeraX-users <chimerax-users@cgl.ucsf.edu> wrote:
>
> Dear all,
>
> Maybe an ignorant or previously-asked question... I believe most people refine cryo-EM models using e.g. Phenix real-space-refine before depositing them to the PDB. Is there a strict reason (practical or philosophical) why cryo-EM models refined in ISOLDE cannot be directly deposited without subsequent refinement in Phenix or Refmac? I have a high-resolution map (2.2 A) but the latter programs seem to actually make my model worse (e.g. Phenix tends to give me rotamers that are not supported by the experimental data). I would prefer to use the model refined in ISOLDE but perhaps I am missing something?
>
> Many thanks for your insight!
>
> Louis
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