Can ISOLDE-refined models be directly deposited to the PDB?
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
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 _______________________________________________ ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/
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#is... 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 _______________________________________________ ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/
_______________________________________________ ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/
-- Altos Labs UK Limited | England | Company reg 13484917 Registered address: 3rd Floor 1 Ashley Road, Altrincham, Cheshire, United Kingdom, WA14 2DT
Hello all, If this is helpful to you, I have been pretty happy using servalcat in the resolution range of 2.5 Å or better (with most side chains resolved). The command "spa_no_refmac" is the one I have been using, making sure to point it to a local copy of the monomer library found here, if not using everything from the CCP4 suite: https://github.com/monomerlibrary/monomers It might still mess up a few things compared to ISOLDE, and ISOLDE doesn't write parameter files for it (yet?), so you might have to read its documentation to figure out the set of options that will refine what you want but no more. The difference map generated by servalcat is very helpful for model building! Cheers, Guillaume On 12/5/25 12:12 AM, Tristan Croll via ChimeraX-users wrote:
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 <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 <mailto: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 <mailto: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 > _______________________________________________ > ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu <mailto:chimerax-users@cgl.ucsf.edu> > To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu <mailto:chimerax-users-leave@cgl.ucsf.edu> > Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/ chimerax-users@cgl.ucsf.edu/ <https://mail.cgl.ucsf.edu/mailman/ archives/list/chimerax-users@cgl.ucsf.edu/>
_______________________________________________ ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu <mailto:chimerax-users@cgl.ucsf.edu> To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu <mailto:chimerax-users-leave@cgl.ucsf.edu> Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax- users@cgl.ucsf.edu/ <https://mail.cgl.ucsf.edu/mailman/archives/ list/chimerax-users@cgl.ucsf.edu/>
Altos Labs UK Limited | England | Company reg 13484917 Registered address: 3rd Floor 1 Ashley Road, Altrincham, Cheshire, United Kingdom, WA14 2DT
VARNING: Klicka inte på länkar och öppna inte bilagor om du inte känner igen avsändaren och vet att innehållet är säkert. CAUTION: Do not click on links or open attachments unless you recognise the sender and know the content is safe.
_______________________________________________ ChimeraX-users mailing list -- chimerax-users@cgl.ucsf.edu To unsubscribe send an email to chimerax-users-leave@cgl.ucsf.edu Archives: https://mail.cgl.ucsf.edu/mailman/archives/list/chimerax-users@cgl.ucsf.edu/
När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/ E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy
Dear all, Thanks for all your quick replies - this is super helpful! Tristan - thank you very much! I didn't know about that tool - I tried and it seems to have worked a treat. At least, my structure changed only minimally during Phenix refinement but the B-factors seem to have been overhauled. I may also compare this servalcat refinement - it sounds interesting! Regarding B-factors - I remember a long time ago there was some discussion about their relevance in cryo-EM: https://www.cell.com/structure/fulltext/S0969-2126(17)30246-0 . I have no idea what the prevailing thoughts in the field are now though! Thanks again, Louis
participants (4)
-
Greg Pintilie -
Guillaume Gaullier -
man6ny＠virginia.edu -
Tristan Croll