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Today's Topics:
1. RSR of tetramer, rotamers of different protomers/chains change
(skaruppan(a)kumc.edu)
2. Re: RSR of tetramer, rotamers of different protomers/chains change
(Pavel Afonine)
3. Re: RSR of tetramer, rotamers of different protomers/chains change
(skaruppan(a)kumc.edu)
4. Re: RSR of tetramer, rotamers of different protomers/chains change
(Pavel Afonine)
5. Re: RSR of tetramer, rotamers of different protomers/chains change
(skaruppan(a)kumc.edu)
6. AmberPrep not prepping (Nikolas)
----------------------------------------------------------------------
Message: 1
Date: Thu, 23 Jul 2026 21:39:03 -0000
From: skaruppan(a)kumc.edu
Subject: [phenixbb] RSR of tetramer, rotamers of different
protomers/chains change
To: phenixbb(a)phenix-online.org
Message-ID: <178484274393.1372232.831230685709650840(a)cci3.lbl.gov>
Content-Type: text/plain; charset="utf-8"
Hi All,
I want to apologize in advance for such a basic question.
I'm working with a tetrameric protein and am attempting to build a
model. The resolution estimate of the map determined by cryoEM is 3.8A.
Some parts of the map are great while some other parts not so much. To
assist with modelling the tetramer, I used the local refinement of one
protomer to build a protomer model (one chain). I then used that
protomer model as a template to build the full tetrameric model, and I
make sure that each protomer is exactly the same (using ChimeraX or
coot) by copying one chain over to the next and making certain they are
all exact copies of one another. When doing real space refinement in
phenix 2.0-5936 of the tetramer model, I end up with good statistics and
what I think is a complete model. RCSB/comprehensive cryoEM validation
also seem to complement my modelling. However upon closer inspection I
noticed that many rotamers across the model are different, for example
Chains A and B will have a Gln in one position, while chains C and D
will have rotamers in totally different positions. These rotameric seem
to happen in substandard regions of map quality.
My understanding is that all the protomers in a C4 channel should look
exactly the same. To attempt to rectify this problem I've made sure that
NQH_flips is off (rotameric changes still happen with many different
residues), as well as tried using reference restraints of my protomer,
or NCS constraints of one protomer (these options results in a
degradation of model quality).
I'm missing or misunderstanding something here (or maybe a combo of
both), and I was wondering if there was any advice from the community on
how to proceed.
Kind Regards,
Seb
------------------------------
Message: 2
Date: Thu, 23 Jul 2026 14:56:11 -0700
From: Pavel Afonine <pafonine(a)lbl.gov>
Subject: [phenixbb] Re: RSR of tetramer, rotamers of different
protomers/chains change
To: skaruppan(a)kumc.edu, phenixbb(a)phenix-online.org
Message-ID: <376df4ab-c7c6-4630-82c3-32aa6e2058aa(a)lbl.gov>
Content-Type: text/plain; charset=UTF-8; format=flowed
Hi Seb,
assuming the map was symmetrized (which is likely at 3.8A), steps are:
0. Get initial model (AphaFold, homologous Xray, etc)
1. Find symmetry of the map;
2. Extract box containing symmetry unique part of the map;
3. Dock your model into the5. Once done building/rebuilding, apply symmetry to the built model to
generate the whole tetramer;
6. Clashes are likely at the interface between symmetry copies;
7. Resolve clashes by iterations of manual rebuilding and refinement.
Make sure you run refinement with NCS constraints, for the tetramer you
will have one master and three dependent NCS copies. This will ensure
that all copies are atom content identical and if superposed, their
superposition rmsd is exactly zero. If that's not the case -- something
must have gone wrong.
8. You are all done -- run final Comprehensive Validation in Phenix GUI
and publish/deposit your model.
If the map was not symmetrized -- genuine structural differences are
expected to be observed between symmetry copies -- do essentially the
same steps except at step #7 do NOT use NCS constraints at all during
refinement.
All 0-9 steps can be done in Phenix GUI or in the command line. This is
one of the tutorial items that we demonstrate at most Phenix user
workshops.
Good luck!
Pavel
On 7/23/26 14:39, skaruppan--- via phenixbb wrote:
> Hi All,
>
> I want to apologize in advance for such a basic question.
>
> I'm working with a tetrameric protein and am attempting to build a
model. The resolution estimate of the map determined by cryoEM is 3.8A.
Some parts of the map are great while some other parts not so much. To
assist with modelling the tetramer, I used the local refinement of one
protomer to build a protomer model (one chain). I then used that
protomer model as a template to build the full tetrameric model, and I
make sure that each protomer is exactly the same (using ChimeraX or
coot) by copying one chain over to the next and making certain they are
all exact copies of one another. When doing real space refinement in
phenix 2.0-5936 of the tetramer model, I end up with good statistics and
what I think is a complete model. RCSB/comprehensive cryoEM validation
also seem to complement my modelling. However upon closer inspection I
noticed that many rotamers across the model are different, for example
Chains A and B will have a Gln in one position, while chains C and D
will have rotamer
> s in totally different positions. These rotameric seem to happen in
substandard regions of map quality.
>
> My understanding is that all the protomers in a C4 channel should look
exactly the same. To attempt to rectify this problem I've made sure that
NQH_flips is off (rotameric changes still happen with many different
residues), as well as tried using reference restraints of my protomer,
or NCS constraints of one protomer (these options results in a
degradation of model quality).
>
> I'm missing or misunderstanding something here (or maybe a combo of
both), and I was wondering if there was any advice from the community on
how to proceed.
>
> Kind Regards,
> Seb
> _______________________________________________
> phenixbb mailing list -- phenixbb(a)phenix-online.org
> To unsubscribe send an email to phenixbb-leave(a)phenix-online.org
> Unsubscribe: phenixbb-leave@%(host_name)s
------------------------------
Message: 3
Date: Thu, 23 Jul 2026 23:43:11 -0000
From: skaruppan(a)kumc.edu
Subject: [phenixbb] Re: RSR of tetramer, rotamers of different
protomers/chains change
To: phenixbb(a)phenix-online.org
Message-ID: <178485019104.1372233.17243617721205818498(a)cci3.lbl.gov>
Content-Type: text/plain; charset="utf-8"
Pavel,
Thank you very much for your reply, I do appreciate it greatly.
I've done steps 0-6. Using the Superpose Models in Phenix I've verified
that for my input model, the chains have RMSD values of 0.00 with each
other (i.e. A-B is 0, A-C is 0, etc).
Then with RSR, I've ensured that the NCS constraints on, and I've made
sure that chain 'A' is the master and 'B-D' are the dependents in the
selection panel, and I hit run. Unfortunately I'm missing something or
there's an issue because I still see rotamer changes between chains.
Using the Superpose Models tool in phenix I see that the RMSD beKind Regards,
Seb
------------------------------
Message: 4
Date: Thu, 23 Jul 2026 16:52:39 -0700
From: Pavel Afonine <pafonine(a)lbl.gov>
Subject: [phenixbb] Re: RSR of tetramer, rotamers of different
protomers/chains change
To: skaruppan(a)kumc.edu, phenixbb(a)phenix-online.org
Message-ID: <91864eae-74b6-447e-a9a7-a21966449cc6(a)lbl.gov>
Content-Type: text/plain; charset=UTF-8; format=flowed
Hi Seb,
At this point, I need to take a close look at your files to diagnose the
issue. Could you please share (off-list) the model before refinement
where the RMSD between copies is exactly zero, and the model after
refinement where these chains do not overlay perfectly?
Also, please send the map, .log, and .eff files.
With these files, I’ll try to replicate your results and explain what’s
going on.
Thanks!
Pavel
On 7/23/26 16:43, skaruppan--- via phenixbb wrote:
> Pavel,
> Thank you very much for your reply, I do appreciate it greatly.
>
> I've done steps 0-6. Using the Superpose Models in Phenix I've
verified that for my input model, the chains have RMSD values of 0.00
with each other (i.e. A-B is 0, A-C is 0, etc).
>
> Then with RSR, I've ensured that the NCS constraints on, and I've made
sure that chain 'A' is the master and 'B-D' are the dependents in the
selection panel, and I hit run. Unfortunately I'm missing something or
there's an issue because I still see rotamer changes between chains.
Using the Superpose Models tool in phenix I see that the RMSD between
chains is slight, ~0.03 to 0.02.
>
> What do you suppose I could be missing?
>
> Kind Regards,
> Seb
> _______________________________________________
> phenixbb mailing list -- phenixbb(a)phenix-online.org
> To unsubscribe send an email to phenixbb-leave(a)phenix-online.org
> Unsubscribe: phenixbb-leave@%(host_name)s
------------------------------
Message: 5
Date: Wed, 29 Jul 2026 17:07:02 -0000
From: skaruppan(a)kumc.edu
Subject: [phenixbb] Re: RSR of tetramer, rotamers of different
protomers/chains change
To: phenixbb(a)phenix-online.org
Message-ID: <178534482294.1372232.18070947457302999779(a)cci3.lbl.gov>
Content-Type: text/plain; charset="utf-8"
Hello Pavel,
As I was writing an email and repeating the steps you outlined above,
the RMSDs became 0. So no Phenix level problem, but a user problem. My
belief is that the NCS constraints were not toggled properly, and that
there was a problem with my input model not being docked correctly.
Model is now finished to my satisfaction, and all chains look the same.
All good now, thank you so much for your help!
-Seb
------------------------------
Message: 6
Date: Sat, 1 Aug 2026 11:26:23 -0400
From: Nikolas <nikolas.capra(a)gmail.com>
Subject: [phenixbb] AmberPrep not prepping
To: "phenixbb(a)phenix-online.org" <phenixbb(a)phenix-online.org>
Message-ID:
<CAEaGD_qXbMEM9YeDJ2jG3eLZPMyrD7Z7iJZb3nutM2XmJna1Yg(a)mail.gmail.com>
Content-Type: text/plain; charset="UTF-8"
Hi everyone,
New to this forum, I usually lurk in the European counterpart, which
makes
me a novice on the many tools of phenix.
I’m trying to refine a low-res structure that has a strong signal in the
active site, for which I modeled a putative reaction intermediate.
However,
this molecule is both linear and long (14C) and thus very flexible, and
only the part nested shows clear electron density, leaving out 4-5
atoms.
When I run refinement, seems like the molecule gets crammed into the
available density and this strains geometry. A clipped version of the
ligand fits the density nicely and doesn’t appear strained. Hence, my
attempt to use Amber on phenix.refine.
AmberPrep gives back an error saying that the file is empty and the
leap.log complains both about the ligand and a distance in the protein.
The
ligand was generated with eLBOW using SMILES and AM QM 1, while the
enzyme
is split in an alpha and beta subunits each monomer due to
autoproteolytic
activation and the in-between is highly flexible and thus absent from
the
model.
chains in the PDB file (e.g. diner with ABCD chains instead of just AB)?
Thanks!
Best,
Nikolas
------------------------------
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