[3dem] [EXT] Re: This is my question to all the members experts and non-experts who are working on membrane proteins in the CryoEM

Mon Apr 8 09:35:17 PDT 2024

Hi Aninda,

regarding to bacterial membrane enzymes that potentially form dimers, with each
dimer having a solved molecular weight under 100
kDa. Despite the inherent challenges posed by their small size, similar cases
have been successfully addressed in cryo-EM studies.

Small membrane proteins (MPs) under 100 kDa present challenges due to their low
natural abundance, high hydrophobicity, and
instability in conformation and composition. Additionally, factors such as low
contrast and a poor signal-to-noise ratio (SNR)
critically impact the quality and resolution of analyses.

In cryo-EM, differentiating protein regions from the background—such as
detergents or lipid nanodiscs in amorphous ice—is crucial.
This differentiation is impeded by the significant solvent volume fraction,
complicating image alignment and 3D reconstruction.

The low molecular mass of small proteins results in fewer electrons scattered,
leading to a weaker signal. The SNR further
degrades when proteins are embedded in thicker ice regions, introducing
additional background noise.

Effective cryo-EM analysis necessitates that proteins be homogeneous and stable
in their conformation to ensure that collected
images represent consistent conformational states, allowing for accurate
alignment and averaging. The variability in particle
orientation and conformation among small proteins often leads to poor
alignment, compromising the quality of the averaged image
and the resolution of the final 3D density map.

Strategic Innovations and Solutions in general:

 * Optimization of Vitrification Conditions: refining the vitrification
processes to control ice thickness and enhance contrast,
   and employing advanced detectors (e.g., K3, Falcon) for optimal results with
phase plate and energy filter.
 * Innovations in Detergents and Nanodiscs: exploring new formulations and
structures to stabilize these proteins within their
   native conformations.
 * Advanced Software and Image Processing Algorithms: utilizing sophisticated
algorithms, including blush regularization in RELION
   5 and non-uniform refinement in CryoSPARC, to enhance contrast and alignment
for accurate 3D reconstructions.
 * Use of Rigid Markers and Grid Improvements: investigating the use of rigid
markers out of membrane can improve the image
   alignment quality of side views lead to an better 3D reconstruction map.
 * Employing grids coated with gold and graphene to reduce beam-induced motion
and interaction issues to have better particle
   distribution in the thin ice area.

For further insights,there is also a review article published last year that
discusses approaches to solving sub-100 kDa membrane
proteins using cryo-EM: https://urldefense.com/v3/__https://doi.org/10.1016/j.jsb.2023.107959__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsLBnz1YTY$ 
[https://urldefense.com/v3/__https://doi.org/10.1016/j.jsb.2023.107959__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsLBnz1YTY$ ].

Best regards,

Lifei

>>> Guillaume Gaullier <guillaume.gaullier at kemi.uu.se> 08.04.2024, 12:47 >>>
Hi Aninda,

Here is a recent review on cryoEM of small proteins:
https://urldefense.com/v3/__https://doi.org/10.1016/j.str.2024.03.003__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsLTZ-jo44$ 
[https://urldefense.com/v3/__https://doi.org/10.1016/j.str.2024.03.003__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgWRzsjVBc3UjwUKC4a_IbFBNlxbLfRt0INvqDSUWJasoXaTuv7SrZlB_$]
It is not specific to membrane proteins, but might answer some of your
questions or give you pointers to look further.

This other article with a systematic exploration of data collection parameters
for GPCRs might be more directly relevant to you,
even though GPCRs are larger than the proteins you describe:
https://urldefense.com/v3/__https://doi.org/10.1038/s41467-021-24650-3__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsL6zBNo9I$ 
[https://urldefense.com/v3/__https://doi.org/10.1038/s41467-021-24650-3__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgWRzsjVBc3UjwUKC4a_IbFBNlxbLfRt0INvqDSUWJasoXaTuv0toUZGm$]

When you don’t get answers on this 3DEM list, I suggest to also search the
archives of the CCPEM
list: https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=CCPEM__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsLkxs5akA$ 
[https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=CCPEM__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgAnd if you still don’t find anything relevant, of course you are welcome to
post your questions to the CCPEM list as well. Some
people follow both lists (I do), but I don’t know how much the two populations
overlap, so for certain questions you might reach
more people with potential answers on the other list.

I hope this helps,

Guillaume

> On 6 Apr 2024, at 22:46, Aninda Dutta <anindadutta86 at gmail.com
[anindadutta86 at gmail.com]> wrote:
> 
> Dear 3DEM list members,
> I have two small bacterial membrane enzymes (sub-50 kDa) that I want to work
with. Both of them are known to dimerize under
> certain conditions and I want to understand the conformational dynamics of
these systems under native lipid environments
> (Liposome, nanodiscs) using CryoEM.
> Regarding that, I have some questions to ask all of you - 
>  1. How are we as a community doing with sub-50 kDa CryoEM without using Fabs
or other nanobodies?
>  2. What are the challenges, and can we come over it?
>  3. Tell me any suggestions or tips you may have before starting this
project.
> 
>  
> Last but not the least, I am open to any interesting collaborations.
>  
> Best,
> Aninda
>  
> ---------------------
> Aninda Dutta
> UVA Chem Graduate Student
> 
> University of Virginia
> Charlottesville, VA
>  
> 
> 
> 
>  
> 
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