Single particle tutorial, bad resolution of Refinement 2 step

[Eugene Baulin]

Hello!

I'm totally new to the field of cryo-EM and now going through the EMAN2 tutorial on single particle analysis with b-gal molecule (https://blake.bcm.edu/emanwiki/EMAN2/Tutorials?action=AttachFile&do=view&target=EMAN2_SingleParticleTutorial.pdf).

I have a problem with the Refinement 2 step (step 12, part 2).

After the first refinement (with the aim of 15A), I was able to get 16.7, 15.8, and 14.4A resolutions, based on a various number of input images (22, 30, or all of them respectively).

But the second refinement (with the aim of 6A) gives me only ~9-10A resolutions and, furthermore, the first iteration is always better in resolution than the following (see the attached report, It took the 14.4A structure as input).

My EMAN2 version:

EMAN 2.91 final ( GITHUB: 2021-03-08 11:36 - commit: 81caed2 )
Your Python version is: 3.7.7

What can I be doing wrong?

Thanks in advance!

Best regards,

Eugene Baulin

[Ludtke, Steven J.]

Usually this is caused by a bad initial model. With B-gal's symmetry and shape, there are a couple of plausible but incorrect initial models which can get things stuck in a local minimum for a while. This is generally diagnosed at the initial model generation stage (step 10). Often it is still possible to refine your way back to the correct structure with the low resolution data, but it can take a dozen iterations or more to do it, so it's usually better to go back to step 10 again.  If you post some screenshots of your refined map we can help diagnose if this is the issue. 

--------------------------------------------------------------------------------------
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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<index.html><converge.pdf><resolution.pdf><resolution_masks.pdf>

[Eugene Baulin]

Thanks a lot!

I returned to step 10 and remade an initial model excluding two bad classes instead of just one. I think it helped since now I could get ~6A on the second iteration of the second (->6A) refinement. 

Although I've got "No valid resolution found" for iterations 3 and 4 (see attachment), the result looks fine to me. Should I be worried?

Best regards,

Eugene Baulin

To view this discussion on the web visit https://groups.google.com/d/msgid/eman2/C4716FB5-E846-4DF1-B554-54EFF207E581%40bcm.edu.

[Ludtke, Steven J.]

No, that all looks good.  The "no valid resolution" just means that there was insufficient sampling, ie - the resolution was likely better than the data could provide. This is common with downsampled data, and just means that its time to switch to the fully sampled data. If you run into that with data at full sampling it may mean you might want to collect at higher mag next time. :^)

--------------------------------------------------------------------------------------
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/CAFtdTFVZ8xWNekF_gcgMoAObGgg%2B-XASEuVzAVjYuPxgD24Vrw%40mail.gmail.com.
<index.html><converge.pdf><resolution_masks.pdf><resolution.pdf>