[ccp4bb] Questions about PanDDA modelling and refining

[Vladyslav Yadrykhinsky]

Hello,

I am learning how to use PanDDA and would appreciate some clarifications on the 
modeling and refining steps.
I realize that maybe some questions will be too obvious, but I would appreciate 
the confirmation of my guesses so I might learn to do it the correct way.

I managed to get to step 13 of the tutorial 
(https://pandda.bitbucket.io/pandda/tutorials.html#) (but with my data).

After step 11 in my pandda-export folder, I have the following files:


File name:
Remarks:
1
ensemble-model.log

2
ensemble-model.pdb
Contains both bound and ground-state
3
ensemble-model-restraints.log

4
ensemble-model-restraints.phenix.params

5
ensemble-model-restraints.refmac.params

6
pandda-input.mtz
Does not contain the "event density" of ligand
7
pandda-input.pdb
Does not contain ligand
8
pandda-model.pdb
Contains bound state of ligand
9
pandda-output.mtz
Does not contain the "event density" of ligand
10
pandda-output-event-001.mtz
Contains the "event density" of ligand

In the file ensemble-model.pdb I see water and ligand on the same spot. My 
protein normally has water in that site, but if the ligand is bound - there is 
no water.

Further modeling is required for my structures and thus I move on to step 13 
where I get confused. I have highlighted in bold the instructions of the 
tutorial.


  1.
Generation of restraints for refinement (giant.make_restraints)
It says: "...The output restraint files can then be fed to giant.quick_refine, 
which then either runs phenix or refmac (see below)."

Should I skip this step as I already have the phenix.params and refmac.params 
files?


  1.
Quick-and-easy refinement (giant.quick_refine)
To make refinement more straightforward, giant.quick_refine can be used to 
refine the models.
A normal refinement will look like

giant.quick_refine input.pdb input.mtz ligand.cif restraints.params

Which files should I use here? The pandda-input.pdb, pandda-input.mtz 
ligand.cif and ensemble-model-refmac.params?
If the .params file is for the ensemble, should I rather use then 
ensebmle-model.pdb, padda-output-event-001.mtz ligand.cif and 
ensemble-model-refmac-params?
If I want to include additional parameterization for the refmac, do I manually 
add instructions in the .params file? For example, if I want to increase the 
weight parameter?
  2.
Splitting the ensemble model (giant.split_conformations)

Should it be done on the output of the step 13.2?

  3.
(Re-)modelling of the bound-/ground-states (coot)
The ground-state conformations should be modelled into a ground-state 
("reference") dataset/map (e.g. "*-ground-state-average-map.native.ccp4" from 
pandda.analyse).

I do not have any file in the format native.ccp4

Should I do it with the file pandda-input.pdb with pandda-input.mtz?
The bound-state conformations of the protein are modeled into the appropriate 
event maps, as in pandda.inspect.
Should I use the files pandda-model.pdb with pandda-ouput-event-001.mtz?
However, if you want to edit both structures simultaneously (e.g. to add a 
molecule that is the same in both states), then this should be done to the 
combined ensemble model.
Should I use ensemble-model.pdb with pandda-output-event-001.mtz?
  4.
Re-merging single-state models for refinement.
After you have re-modelled the bound- and ground-states of the protein, you 
need re-assemble the ensemble model for refinement.

giant.merge_conformations ground-state.pdb bound-state.pdb

Maybe it will be more clear which exact files to use here once I go through 
steps 1-4, but if there is something I should be aware of during this step - 
please let me know.

Please let me know if you any of my questions should be clarified more.

I will greatly appreciate any help I can get with this!

Best regards,
Vladyslav





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[ccp4bb] ZipperDB database access issue

[Das, Abhinaba]

Dear Community,

There is this nice web server, ZipperDB 
(https://services.mbi.ucla.edu/zipperdb/intro), that predicts the fibrillogenic 
propensity of a given peptide segment. However, it seems to have been down for 
a couple of months now. I have emailed them a few times but have not received a 
response, likely because the email is not monitored regularly. Has anyone else 
experienced this issue or knows anything about what is going on?
Thanks,
Abhi





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Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era?

[Javier Gonzalez]

Thank you all for your thoughtful answers.

I'll stick to the crystallographic data and model as much as I can with
reasonable occupancy and B factors, even if the result is a truncated model.

I don't believe in the zero occupancy trick either. I just thought the PDB
was more flexible these days, since the community of structural biologists
seems to have accepted the highly accurate and highly imprecise coordinates
of AF-generated noodle-like loops in the UniProt database.

It sounds like the PDB-DEV database is the right place to deposit those
hybrid models I had in mind, and leave the genuine crystallographic models
for the PDB.

Best wishes,
Javier

On Sun, Jul 28, 2024 at 2:52 PM Guillaume Gaullier <
guillaume.gaull...@kemi.uu.se> wrote:

> Hello Javier,
>
> Placing atoms implies that you know they are present somewhere (possibly
> with some uncertainty on exactly where), but setting their occupancies to
> zero implies that you know they are nowhere at all. This is a paradox.
>
> I think atoms with zero occupancy make no sense in a final deposited model
> (they could be useful as a working intermediate to exclude the bulk solvent
> model, but this is unrelated to what you describe).
> So in this particular case, partial occupancies only make sense for
> multiple conformations (and should add up to 1), as Pavel describes.
>
> If you can’t resolve more than one conformation, maybe a better approach
> is to fix the coordinates to what AlphaFold suggested (which is often
> reasonable, but check their pLDDT to assess this) and refine to let the
> B-factors of these atoms rise. This will convey the large uncertainty on
> their positions. I think it is a valid approach because you know these
> residues are there somewhere (in other words, to me you would need evidence
> of their absence to justify truncating these loops: SDS-PAGE showing that
> the protein is cleaved, for example).
>
> I hope this helps,
>
> Guillaume
>
> On 28 Jul 2024, at 17:32, Pavel Afonine  wrote:
>
> 
>
> Javier,
>
> Flexible loops may be better modeled with ensembles of N models, meaning
> the occupancy of each-one would be 1/N, and the map contours to visualize
> them should be chosen as 1/N sigma (not 1 sigma). While model prediction
> tools such as AlphaFold are helpful, they don't suddenly lift the
> requirement for the atomic model you release to the world to fit the
> experimental data! With this premise in mind, the approaches to validate
> your model geometry and model-to-data fit quality have not changed before
> and after the AlphaFold era.
>
> Whether you truncate residue side chains/loops that you don't see or keep
> them with zero occupancy is a perennial question on this list that has been
> coming up for decades, and I have yet to see an answer that everyone agrees
> on!
>
> All the best,
> Pavel
>
>
> On Sun, Jul 28, 2024 at 8:13 AM Javier Gonzalez  wrote:
>
>>
>>
>> Dear CCP4bb,
>>
>> I'm refining the ~3A crystal structure of a big protein, largely composed
>> of alpha helices connected by poorly-resolved loops.
>> In the old pre-AlphaFold (AF) days I used to simply remove those
>> loops/regions with too high B factors, because there was little to none
>> density at 1 sigma in a 2Fo-Fc map.
>> However, considering that the quality of a readily-computable AF model is
>> comparable to a 3A experimental structure, and that the UniProt database is
>> flooded with noodle-like AF models, I was considering depositing a combined
>> model in the PDB.
>> Once R/Rfree reach a minimum for the model truncated in poorly resolved
>> loops, I would calculate an augmented model with AF calculated missing
>> regions (provided they have an acceptable pLDDT value), assign them zero
>> occupancy, and run only one cycle of refinement to calculate the formal
>> refinement statistics.
>> Would that be acceptable? Has anyone tried a similar approach?
>> I'd rather do that instead of depositing a counterintuitive model with
>> truncated regions that few people would find useful!!
>>
>> Thank you for your comments,
>>
>> Javier
>>
>> --
>> Dr. Javier M. González
>> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
>> Universidad Nacional de Santiago del Estero (UNSE)
>> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
>> Santiago del Estero. Argentina
>> Tel: +54-(0385)-4238352
>> Email  Twitter 
>>
>>
>> --
>>
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> När du ha

[ccp4bb] Biological Cryo-EM Expert Position - UC Irvine

[Celia Goulding]

Dear All 

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It is a great position, so please apply.

Celia.



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