[phenixbb] Problems with sharpening in Autosharpen

Carlos HENRIQUE FERNANDES carlos.henrique_fernandes at sorbonne-universite.fr
Tue May 25 09:34:39 PDT 2021

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, 

De: "Tom Terwilliger" <tterwilliger at newmexicoconsortium.org> 
Para: "Carlos HENRIQUE FERNANDES" <carlos.henrique_fernandes at sorbonne-universite.fr> 
Cc: "phenixbb" <phenixbb at phenix-online.org>, "Tom Terwilliger" <tterwilliger at newmexicoconsortium.org> 
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 < [ mailto:carlos.henrique_fernandes at sorbonne-universite.fr | carlos.henrique_fernandes at sorbonne-universite.fr ] > 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, 
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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 
Email: [ mailto:tterwilliger at newmexicoconsortium.org | tterwilliger at newmexicoconsortium.org ] 
Tel: 505-431-0010 

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