I’ve been working on reconstructing amyloid fibers and noticed some reluctance in the community about using CryoSPARC for this purpose. Some reviewers/researcher in this field have even suggested that other software packages might be more suitable for solving these types of structures. I wanted to kindly ask if you might have any insights into the reasons behind this hesitation in using CryoSPARC for this purpose, or if it’s just a field-bias issue.
Additionally, if anyone you know has tips or tricks for handling amyloid fiber reconstructions, I would greatly appreciate any advice you can share. I’m particularly interested in improving the quality of my reconstructions and learning more about the nuances of different software options for these challenging datasets.
Thank you in advance for your time and assistance!
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It sounds like you are asking, “should I use cryosparc for helical reconstructions?”
If yes, then there are few approaches, but helical reconstruction is highly error prone if parameters are not correct - it is easy to get wrong.
The RELION lab https://www2.mrc-lmb.cam.ac.uk/group-leaders/n-to-s/sjors-scheres/ does much work on similar fibrils in addition to all of the software development. They have some systems were BLUSH regularization helps for similar systems.
Before getting there I would ensure you are using all of the tools for picking. The Filament Tracer https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/helical-reconstruction-beta is a great start.
A big point is that the deep pickers (CrYOLO and TOPAZ) can do a great job picking repeating units on filaments if you know what to pick.
It could be worth to to explore beyond single particle. Sub-tomogram averaging has also been used with success in such systems. If you have a novel amyloid, AFM and ssNMR could also provide novel insights.
Hi Mark,
Thank you for your reply, but it wasn’t exactly my point. I was curious why does the Amyloidal-related community don’t use CS as an standard workflow for Helical reconstruction. I’m aware of the other packages and, indeed, Relion is responsible for almost 100% of EMPIAR/EMDB depositions.
So, I was just curious to know why is this community reluctant against CS as an useful tool for this type of data processing. For example, I was able to only find a single manuscript that uses only CS for it (https://www.pnas.org/doi/10.1073/pnas.2406775121).
It was more philosophical than technical question.
Thanks
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Understood. I think the whole thing with Sjors then. He has done excellent work on such systems and also develops RELION.
can also see https://github.com/asarnow/pyem/wiki/Export-from-cryoSPARC-v2-and-later. “Why do Relion and cryoSPARC results look different?” from @DanielAsarnow
It could be interesting to see if you can take an EMPIAR data sets and where it goes with CryoSparc vs. RELION or something else (SIMPLE 3.0, cisTEM, Thunder, …very long list). The general advice is to try as much as you can.
In fact we did it, and some of Relion-published EMDB entries are not reproducible. It’s interesting how some reviewers write in between lines pretty much saying "if you haven’t use Relion, it’s incorrect or unacceptable ".
Then, I was curious why amyloidal-field is “too biased” towards a single package and way of doing it.
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My understanding is that helical processing in CS is not yet optimized for dealing with amyloids, which have some very specific characteristics - small rise and small twist most particularly.
Sjors and others have put a lot of effort into developing protocols to use RELION to process such cases, and added a number of amyloid specific tools/scripts, whereas nothing comparable yet exists for CS.
Doesn’t mean you can’t use CS for amyloids though! Just that the tools are not as well-developed for that particular use case yet.
Re reviewers complaining, the density should speak for itself. If the density map (and model fit) is unambiguous, it should not matter what software package you used.
EDIT: Job: Helical Refinement | CryoSPARC Guide - note difficulties with amyloid fibrils are pointed out explicitly in the CS guide.
Hi @olibclarke,
I completely agree with your final statement, and this is something that we try to argue making the comment that it shouldn’t matter, but I think the common sense in the field is: if it’s not done using Relion, this is non-acceptable.
Anyway, thank you and @Mark-A-Nakasone for the insights.
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