Working with very small protein

[Dmitry]

Dear colleagues,

I am dealing with very small protein.
Could you please provide recommendations for image processing?
Currently, I am curious about the particle picking // 2D and 3D refinement.

Thank you.

Kind regards,
Dmitry

[DerLorenz]

Hi Dimitry,

Here is a tutorial for the processing of membrane proteins:

Tutorial: Tips for Membrane Protein Structures | CryoSPARC Guide

The settings explained there helped me with small non-membrane targets before. Alternatively, this issue has been discussed elsewhere. The approaches suggested in these threads also helped me to get a grip on smaller targets.

Small membrane proteins: 2D classification and ab-initio reconstruction

Hi @M.Grieben, Thanks for your post - this is an interesting case. The parameters you chose for 2D classificaiton are appropriate for the context. The batchsize-per-class can help here, and in fact you can turn it up to a very large value (say 1000). This will make the job slower, but it will ensure that the signal from more particles is available at each iteration of classification. You can also play with the “Initial classification uncertainty factor” - a larger value will cause classificat…

Cannot align small protein complex particles

Hi , I’m facing more or less the same problem. I’ve collected a dataset of a small and elongated protein complex (~75 kDa) with good contrast too. We align the 2D but we have issues with the 3D. I attach a pair of micrograps for reference: [Screenshot 2020-07-31 at 11.52.36] [Screenshot 2020-07-31 at 11.54.12] They were collected on a Talos Arctica with a K2 at 36 K magnification (1.2 A practical, 1.13 A corrected). One shot per hole on a 1.2/1.3 Au grids. Defocus was settled …

Cheers,
Lorenz

[mannda]

If you want to train with a positive control, we uploaded micrographs of 65 kDa hemoglobin that lead to a 3 ang map to EMPIAR a while back EMPIAR-10721 CryoEM single particle analysis of 65 kDa human haemoglobin using a 200 kV Talos Arctica

In our hands increasing the dose experimentally to 60-80 e/ang2 had higher impact than processing tricks

[ctueting]

Hi Dmitry,

do you “see” your particle in the micrograph? If not, do you have CLEM or phase-plate contrasted information, where your small protein is actually in the micrograph?

IF you get the right locations unambigously, it should be possible, to distinguish signal from noise.

Best
Chris

[Dmitry]

Hi Chris,

Though I do appreciate your suggestions on the applicability of CLEM or phase-plate, my initial inquiry dealt with the issues of image processing analysis. Well, I appreciate your contribution. However, image analysis Specialists have already provided the working solution above that solves my primary question.

Thanks for joining the conversation.

As always,
Dmitry

[roney.tucker]

I have protein with 40 kD and getting difficult to reconstruct the particles as it is very tricky to pick particles. HoweverI was able to get 3.5 A map, but the particle was borken in the map. I am still struggling to get good resolution.

[Dmitry]

Hello @roney.tucker. Yes, indeed, I have a similar situation. Currently - struggling with particle picking. Great to know that you managed to push it for 3.5A. This is fantastic. What particle picking tool worked for you best? Kind regards, Dmitry