Box Size Error- Negative stained Dateset

[Sahar NH]

Hello Eman2 Team,

I am trying to familiarize myself with Eman2. I have negative stained micrographs and less than 2000 particles.

I followed the tutorials till Auto CTF, it fails with the following error:

The length of your particle on its longest axis should be <= 129.6. If your particle is larger than this, you either need a larger box-size, or you will need to proceed with CTF fitting manually (and will likely achieve suboptimal results).

I also tried different box size but the error persists. Then I tried Manual CTF fitting and followed with Auto CTF, it did not resolve it.

I have attached an screenshot that might help with the situation.

Can you help with this?

Many thanks for any help and your attention.

Best,

Sahar

[Steve Ludtke]

Hi Sahar,

that message is printed after the program successfully completes. It isn't saying that your particle IS too large. At this point in processing the software has no idea how large the particle actually is. This message is simply a reminder to make sure your box size is sufficient because going back and changing the box size after this point in processing can be challenging.

Based on the visual size of your particles I'd say the box size you are using right now is significantly too large. The target should be a box size between 1.5 and 2x the longest axis of your particle. This works on both ends. If the box size is more than 2 or maybe 2.5x the size of the particle, processing won't work very well.

Looking at your data, I'm afraid you are headed for a disappointing reconstruction as there is clearly a lot of incompatible size heterogeneity in your particle population, and the particles themselves appear to be a single amorphous blob. If you continue (with a smaller box size), assessing things at the 2-D class average stage is going to be critical in determining whether there is anything useful there to try and reconstruct...

cheers

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