Electron Microscopy and Image Processing

Hello

I wished to try b-factor sharpening of my reconstructions using the bfactor program, but do not know how to judge parameters such as resolution range, filter radius etc for the software’s inputs.

Could I please be advised as to what would be appropriate values, in light of things such as particle size, resolution as per the various criteria, the Nyquist etc.?

Please also let me know as to whether the models ought to be binary masked, and if the technique works with negative-stain models as well.

Hi,
I do not want to reinvent the wheel: Would anybody give away a script that generates a number of combinations of random angles to be used for making random projections of a volume?
Thanks, D.

Hi,

What's the best resolution range to use for obtaining a good B-factor estimate (bfactor v1.04)?
In my case, the structure is 13A (0.5 FSC cutoff) or 11A (0.143 FSC cutoff).

Is there a good way to determine if the B-factor value that you are using is too high (or low)?

Thanks for the help.
Cheers,
Ernesto.

Leginon and FEI's EPU have drift control built in their applications. In SerialEM, there is no specific command or function for drift control. However, using Macro, it can be done easily. Here I show macro example how we do it.

http://www.bio.brandeis.edu/KeckWeb/emdoc/en_US.ISO8859-1/articles/Seri…

In single particle application, we take a lot of images. And for CTF flattening purpose, we want to change the defocus a little bit. With SerialEM, this can be done super easy - we simply change target defocus before doing autofocus. Here is an example showing how powerful and flexible the macro can provide.

http://www.bio.brandeis.edu/KeckWeb/emdoc/en_US.ISO8859-1/articles/Seri…

The goal is simple: you want to have multiple montage maps collected. For example, you found a few "good" meshes at LM mag, and you want to have maps for all
of them at a mag say 3000X. I have found this could be troublesome for a new SerialEM user, I hope this FAQ can help them.

http://www.bio.brandeis.edu/KeckWeb/emdoc/en_US.ISO8859-1/books/faq/boo…

Moving stage Z is perhaps more challenging than X,Y. But it is a very reasonable request to ask specimen to move to Eucentric Height at each location you want to record the final image. With SerialEM, it can be done relatively easy. There is still a room to improve the accuracy, taking into account the facts like normalize objective lenses etc.. Here I am trying to explain how it is done.

http://www.bio.brandeis.edu/KeckWeb/emdoc/en_US.ISO8859-1/articles/Seri…

Hi All,

First of all thanks for any answer.

My question could seem silly but I cannot work it out. Basically, does a median filter affect the final resolution?

I am working in stain and boxing particles. I am using a median filter with kernel 5x5 in order to reduce noise and relief my eyes.
As expected after a median filter is applied, the edges look better. I was wondering whether those particles can be used for image processing and the median filter can help in aligning particles usless it does not affect the resolution.

Thanks

goue

We had discussion before using hole template for stage positioning. It works well in most cases. However, there are situations which this might not work so well. For example, when there is no regular hole on the grid, or we do not want every hole. In some cases, we can see the sample position in hole like with filament sample which may not be quite in the center of the hole. In negative stain case, there is no hole.

When trying to plot my FSC using PLOTALL in imagic i get the following error message

forrtl: severe (24): end-of-file during read, unit -5, file Internal List-Directed Read

Anybody know what the problem might be? Is there better programs for this?

Many thanks

Jimmy