Hello,

 

preferential views can give quite significant distorsions in cryo-EM maps.

What is your current Euler angle distribution plot?

If preferential views are significant and/or if you see fine stripes in the sections of your 3D reconstruction you can try to remove some of the most prominent preferential views (i.e. remove those particles), but be careful to keep the more rare views (these tend to get removed because the quality of re-projections is not as good / is more noisy, which may disturb structure (cryo-EM map) refinement).

 

Best,

 

Bruno

 

 

From: [email protected] [mailto:[email protected]] On Behalf Of Carlos HENRIQUE FERNANDES
Sent: 25 May 2021 18:35
To: Tom Terwilliger <[email protected]>
Cc: phenixbb <[email protected]>
Subject: Re: [phenixbb] Problems with sharpening in Autosharpen

 

Hi Tom,
Thanks for the message.
I did the tests that you suggested to me.

I runned the autosharpen tutorial data supplied with Phenix and it worked normally. I blurred that map to 300 A**2 and the resulting map was indeed similar to my map. Finally, I applied the Autosharpen to the blurred map and it was applied a B-sharpen of 222.44 A**2. So, everything worked normally.

I was thinking that may be disturbing the sharpening is that I have a clearly preferred protein orientation in single-particle analysis. In my data set, the side views are more abundant than the rest of views in my 2D classes (and the number of particles in side view 2D classes are much more larger than in the rest of views 2D classes).  What do you think?

Thanks again for the help.
All the best,
Carlos.

De: "Tom Terwilliger" <[email protected]>
Para: "Carlos HENRIQUE FERNANDES" <[email protected]>
Cc: "phenixbb" <[email protected]>, "Tom Terwilliger" <[email protected]>
Enviadas: Sexta-feira, 21 de maio de 2021 22:16:57
Assunto: Re: [phenixbb] Problems with sharpening in Autosharpen

 

Hi Carlos,

Normally if you apply a B-sharpen of 220 to a 3 Å map, the new map should look really different from the original map (no matter whether you do it yourself or autosharpen does it).  So best guess is that something is not happening the way you would like it to be.

 

A suggestion:  run the quick autosharpen tutorial data supplied with Phenix.  Presumably it will work normally.  Then tell autosharpen to blur that map to 300 A**2.  This map should look something like your map (does it?). Now apply autosharpen to the blurred map.  It should apply a B-sharpen of about 250 or so (does it?).  And the new blurred - then - sharpened map should look good.

 

Let me know if that doesn't work!

All the best,

Tom T

 

On Fri, May 21, 2021 at 8:50 AM Carlos HENRIQUE FERNANDES <[email protected]> wrote:

Dear all,

I am facing some problems with sharpening in Autosharpen.
My current map is about 3.2 Å resolution, which I obtained after CTF-Refinement and Polishing in Relion.
When I use Autosharpen in default parameters, it tells me that are applied an overall Bsharp applied of 220.34 and a final Biso of 87.42; but very little sharpening is done; I was unable to observe a typical helices sharpening at this value of resolution.
I did some tests setting progressive Bsharp values (240; 260; 280; 300), but I am still unable to visualize a typical helices sharpening.
Has anyone had problems like that? Any tips?
Thanks in advance,
Carlos.

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--

Thomas C Terwilliger

Laboratory Fellow, Los Alamos National Laboratory

Senior Scientist, New Mexico Consortium

100 Entrada Dr, Los Alamos, NM 87544

Tel: 505-431-0010