bad particle outputs

[ap...@pdx.edu]

Here is my info:

EMAN 2.91 final ( GITHUB: 2021-03-08 12:05 - commit: 81caed2 )

Your EMAN2 is running on: Mac OS 10.16 x86_64

Your Python version is: 3.7.9

I am working with a few negative stain data sets and am getting funky results for my particle outputs (see "bad" attachment). I have processed negative stain data in EMAN for quite some time, but haven't had this issue before. I processed this data in EMAN 2.22 (on a different computer) and get the correct results (see "good" attachment). This has happened one other negative stain data set as well, but I don't understand why, and most data sets turn out normal. 

My work flow is pretty basic and use defaults: evaluate and import -> interactive particle picking -> manual ctf processing (auto fitting, structure factor, redo auto fitting, generate output) -> build sets. Looking at the CTF fits, they all look normal.

Any feedback would be great. Let me know if you need more information.

Adam Miller

[Ludtke, Steven J.]

You mean class-averages?  What do you mean by "funky"?  Are you referring to the apparent blurriness of the class-averages or something else?  In principle I don't see anything wrong with either image. It looks like there may have been a bit too much amplitude contrast correction in the first image, but otherwise seems ok?  Did you follow the tips here:

http://eman2.org/NegativeStain

?

--------------------------------------------------------------------------------------
Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)

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<bad.png><good.png>

[ap...@pdx.edu]

These are not class averages, the are particle images. Both images I attached (bad and good) are from the same data set and the screenshots were taken when I look at the particles in my .lst (opening them with the filter tool). Yes, I did follow the negative stain suggestions from that link. I suppose I am calling the "bad" particles funky because they are nothing like what I expect (i.e., what is seen in the "good" image). I have worked with the protein in negative stain for a long time so I know what I expect to see.

Adam

[Ludtke, Steven J.]

Sorry, completely unclear what stage of processing you're at. If you are just talking about raw particles after boxing them out with boxer or e2boxer, then there should be absolutely no difference between any versions of EMAN1 or EMAN2. It is simply extracting raw particle values from the micrographs. If you ran CTF autoprocessing on the particles, and we are looking at particles after CTF phase flipping and/or filtration, then it depends on what version of the particles you are showing. Following the normal processing pipeline, you might have something like:

BGal_000175__ctf_flip_fullres.hdf

BGal_000175__ctf_flip_invar.hdf

BGal_000175__ctf_flip_lp12.hdf

BGal_000175__ctf_flip_lp5.hdf

BGal_000175.hdf

the non __ctf_flip files should contain particle data exactly as it is in the raw micrographs...

--------------------------------------------------------------------------------------
Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)

To view this discussion on the web visit https://groups.google.com/d/msgid/eman2/74759937-e670-48d3-b9da-f86ff4e0fb4en%40googlegroups.com.

[ap...@pdx.edu]

I believe I figured out the problem. In the ctf generate output tab I have to uncheck the "snrfilt" option and then I get the correct looking particles.

[Ludtke, Steven J.]

Sure, just depends on what you're after. However, it's true that SNRfilt isn't as useful for stain data, which already has high contrast at low resolution.

--------------------------------------------------------------------------------------
Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)

To view this discussion on the web visit https://groups.google.com/d/msgid/eman2/e210ecfd-7c91-4d94-8a73-3ebe22d19434n%40googlegroups.com.