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CCPEM  March 2016

CCPEM March 2016

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Subject:

Re: Artifacts in the map (due to misaligned particles?)

From:

Gökhan Tolun <[log in to unmask]>

Reply-To:

Gökhan Tolun <[log in to unmask]>

Date:

Thu, 31 Mar 2016 12:18:25 -0400

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (88 lines)

Thanks for your suggestion Giulia. I tried 25A, and it seems to have worked
as both the artifacts seem to be gone, and the resolution has improved
somewhat.
Gökhan

-----Original Message-----
From: Collaborative Computational Project in Electron cryo-Microscopy
[mailto:[log in to unmask]] On Behalf Of Sjors Scheres
Sent: Thursday, March 31, 2016 1:53 AM
To: [log in to unmask]
Subject: Re: [ccpem] Artifacts in the map (due to misaligned particles?)

Hi,
I think Giulia's suggestion is a good one. Sometimes at low-res the
orientations aren't well defined and starting from a high-res reference may
help. As always: just maintain your own critical judgement regarding
overfitting and final resolution estimates.
HTH,
Sjors

> Dear Gökhan,
>
> I had a similar issue. I didn't see any clear X-shaped artifacts but 
> the map clearly wasn't right. The way I noticed was by running 3D 
> classification from the refined particles, without allowing for 
> alignments, and I got classes that were clearly the same molecule 
> rotated. You might want to try this to see if this really is your problem.
>
> The way I solved it was by increasing the low-pass filter for the 3d 
> refinements to 20A. Even a 40A filtered map was too "pseudo-2-fold" 
> for the aligments to work, whereas the 20A-filtered one worked
beautifully.
> You might need to find the coarser possible filter that eliminates the 
> error - this will depend on the size and shape of your object.
>
> As long as the final resolution goes significantly beyond your initial 
> filter I think you'll be fine - although I'd be very interested in 
> hearing people's comments regarding this.
>
> Giulia
>
> On 28/03/2016 23:55, Gökhan Tolun wrote:
>>
>> Hi,
>>
>> I am working on two projects with no symmetry, and in the maps 
>> generated by the refinement process I see artifacts that look as if 
>> some of the particles were misaligned 180 degrees in one axis, 
>> creating a pattern in the 3D reconstruction slices that looks like 
>> two densities crossing each other (with a general X or + appearance 
>> at the points where the two densities meet). Also, if I move through 
>> the slices of the reconstructed 3d volume (e.g., using 3DMOD), the 
>> density continuity moves in two different axes that are 90 degrees to 
>> each other (best-effort explanation), so I am quite sure that there 
>> is something wrong.
>>
>> I do not see any relevant parameters I can play with in the GUI, and 
>> when I looked at the relion_refine parameters from the command line, 
>> nothing obvious caught my eye that is listed as may be related to 
>> such an issue.
>>
>> So, is there a way to avoid misalignment of particles during a C1 
>> refinement? May any of the “expert” or other command-line parameters 
>> be fiddled-with to fix such a faulty behavior during a 3D refinement, 
>> and if so, which ones and how?
>>
>> Thanks.
>>
>> Gökhan
>>
>
> --
> Giulia Zanetti
> ISMB, Birkbeck College
> Malet St. London
> WC1E 7HX
> 02076316898
>
>


--
Sjors Scheres
MRC Laboratory of Molecular Biology
Francis Crick Avenue, Cambridge Biomedical Campus Cambridge CB2 0QH, U.K.
tel: +44 (0)1223 267061
http://www2.mrc-lmb.cam.ac.uk/groups/scheres

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