[3dem] A strong density in the cryo-EM map

[Dr. Alexander Belyy]

Dear colleagues,

I would like to thank you all for all the useful suggestions.

We tried to do an elemental analysis with ICP-MS, but it only gave 1 molecule of zinc for 2 molecules of our protein. We will repeat the experiment.

I have checked several cryo-EM models with IP6. Indeed, it has a similar size and coordination, but IP6 is rather flat, while our mysterious density is more pyramidal and hollow inside.

Unfortunately, the AlphaFill prediction did not yield any hits.

The UV-Vis and excitation spectra showed no special peaks. I will return to this if I manage to obtain a mystery density-free protein mutant to serve as a control.

Chelating with EDTA or EGTA did not change the shape of the protein in negative stain EM and had no effect on thermal stability. We have not yet attempted to obtain a cryo-EM reconstruction in the presence of a chelating agent.

The ideas of trying a machine learning algorithm https://urldefense.com/v3/__https://academic.oup.com/bioinformatics/article/35/3/452/5055122__;!!Mih3wA!AD8wSs8q6Z4d2pqYrsiwQvK5U9Wk0rdzkI5860HuWPp7Ke9rq6k1gOdNg_R-9A_jEpZoC1bFkT1rtDoz3VoYLGDTpMGSm65fqfarp880$  and checking if the mass is radiation resistant were very good. I will give it a try.

The protein was purified from E. coli.

Many thanks to all of you!

Best regards,
Alexander



________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Dr. Alexander Belyy <alexander.belyy at mpi-dortmund.mpg.de>
Sent: 17 January 2023 11:17
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] A strong density in the cryo-EM map


Dear community of structural biologists,

In a 3.5 Å cryo-EM reconstruction, we have discovered a large density of 10-12 Å diameter that we are having difficulty identifying (see attachment). This density is surrounded by 5 arginines, 3 asparagines, 2 glutamines, 1 aspartate, and 1 glutamate coming from 4 different regions of the protein. Functionally, the density is needed to hold the protein together. This density appears at higher thresholds than the protein itself and is visible even at the 2D class level. After local refinement and sharpening, the density becomes pyramid-like with a series of smaller spheres with pseudosymmetry (or C3?).

We would be grateful for any suggestions as to what this might be.

Thank you!

Best regards from Dortmund,
Alexander
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