[3dem] 3dem Digest, Vol 130, Issue 38

Tue Jun 26 02:01:53 PDT 2018

Hi Lu,

I actually have this problem as well, the pre-tilt angle of the CTF mrc
file is opposite to the FFT of subtomogram.

Normally it doesn't matter a lot, but if trying to get something relatively
high resolution, it will cause some problem... on the other hand, if the
pretilt angle is quite high, it will also may cause some trouble...like
elongated structure.  I normally just flip the sign of the order file
before create the CTF files, wondering if there is other options.

Best
Qiang

2018-06-26 6:01 GMT+02:00 <3dem-request at ncmir.ucsd.edu>:

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> Today's Topics:
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>    1. Re: Missing wedge masking in RELION (Lu Gan)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 26 Jun 2018 11:59:46 +0800
> From: Lu Gan <lu at anaphase.org>
> To: "Gibson, Kimberley" <kimberley.gibson at yale.edu>
> Cc: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Missing wedge masking in RELION
> Message-ID:
>         <CAPbw-UmN=YRETYQiU5vMrhnM2nprDpYqc+xLgEAyCGv8akOyhQ at mail.
> gmail.com>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Kimberley and Steiner,
>
> After reading your messages, I looked at some of our old runs. Many of our
> tomograms have a "pre-tilt" relative to the tilt axis, i.e., the missing
> wedge is rotated either clockwise or anticlockwise around the tilt axis. I
> noticed that for one of our runs, the RELION _ctf.mrc file was pre-tilted
> in the opposite direction relative to the tomo (see attachments). You can
> see this effect if you open the _ctf.mrc file in IMOD, then flip it around
> the X axis so that you're viewing down the tilt axis, then compare with the
> flipped tomogram viewed as a FFT. In principle, this mismatch can make
> unmeasured parts of a particles' Fourier transform upweighted and the
> measured parts downweighted. After digging through the auxiliary files, I
> found that the tomogram in question had a .tlt file in which the angles
> were ordered from positive to negative. Our other tomograms had .tlt files
> with angles ordered negative to positive: the _ctf.mrc files in these runs
> matched the respective missing wedges. I did not see any difference in my
> subtomogram averages after I corrected this pre-tilt mismatch, but the
> averages might not have enough resolution to show any artifacts. I'd be
> curious if anyone else has had this pre-tilt mismatch problem and if
> correcting for the .tlt file made a difference.
>
> RELION _ctf.mrc file:
>
> FFT of tomogram:
>
>
>
>
> Cheers.
> Lu
>
> --
> Lu Gan
> Assistant Professor
> Department of Biological Sciences
> Centre for BioImaging Sciences
> National University of Singapore
> 14 Science Drive 4
> S1A, Lvl 2
> Singapore 117543
>
> www.anaphase.org
>
> Tel: (65) 6516 8868
> Fax: (65) 6776 7882
>
> On Fri, Jun 8, 2018 at 8:46 PM, Gibson, Kimberley <
> kimberley.gibson at yale.edu
> > wrote:
>
> > Hi Sjors,
> >
> >
> > Thank you for the really helpful update. That does sound very promising.
> >
> >
> > Best,
> >
> >
> > Kimberley Gibson
> >
> > Research Associate, Charles Sindelar Lab
> >
> > Dept. Molecular Biophysics and Biochemistry
> > Yale University
> >
> > ------------------------------
> > *From:* Sjors Scheres <scheres at mrc-lmb.cam.ac.uk>
> > *Sent:* Thursday, June 7, 2018 3:25:24 PM
> > *To:* Gibson, Kimberley
> > *Cc:* Steinar Halldorsson; 3dem at ncmir.ucsd.edu
> > *Subject:* Re: [3dem] Missing wedge masking in RELION
> >
> > Hi Kimberley, Steinar,
> > The way this is done is published: see Bharat et al, Structure 2016.
> > Kimberley, yes you are right, and we are on this one! We are working with
> > the Briggs group to generate better 3D CTFs for subsequent use in relion
> > refinement and classification. Hopefully more about this some time in the
> > medium-future.
> > HTH,
> > Sjors
> >
> >
> > >>From personal experience, after using RELION to perform subvolume
> > >> averaging on a structure with an extreme preferred orientation - that
> > >> even tomography would struggle to overcome - I found that RELION did
> > tend
> > >> to overcompensate the missing wedge. Upon looking at the 3D FTs of our
> > >> averaged structure, we noticed that much of the Fourier space was
> filled
> > >> in and we were rather optimistic about the results. I would approach
> > >> these results a little more cautiously now as I believe that RELION
> was
> > >> blurring together certain features which are now more resolvable using
> > >> other programs that do apply wedge masking. This is something that
> could
> > >> be discussed for future updates to RELION and incorporated into the
> > >> pipeline, hopefully.
> > >
> > > Sincerest regards,
> > >
> > > Kimberley Gibson
> > >
> > > Research Associate, Charles Sindelar Lab
> > >
> > > Dept. Molecular Biophysics and Biochemistry
> > > Yale University
> > >
> > > ________________________________
> > > From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Steinar
> > Halldorsson
> > > <steinar.halldorsson at crick.ac.uk>
> > > Sent: Wednesday, June 6, 2018 5:32:58 AM
> > > To: 3dem at ncmir.ucsd.edu
> > > Subject: [3dem] Missing wedge masking in RELION
> > >
> > >
> > > Dear Tomography folks
> > >
> > >
> > > A question regarding sub-tomogram averaging in Relion, how does the
> > > software treat the missing wedge? Wishful thinking, but does it in any
> > way
> > > automatically apply a mask to the individual particles? Is there a way
> > (or
> > > will there be a way in the next versions) to apply such a mask?
> > >
> > >
> > > Many thanks,
> > >
> > > Steinar
> > >
> > > The Francis Crick Institute Limited is a registered charity in England
> > and
> > > Wales no. 1140062 and a company registered in England and Wales no.
> > > 06885462, with its registered office at 1 Midland Road London NW1 1AT
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> >
> >
> > --
> > Sjors Scheres
> > MRC Laboratory of Molecular Biology
> > Francis Crick Avenue, Cambridge Biomedical Campus
> > Cambridge CB2 0QH, U.K.
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-- 
Qiang (John) Guo
Department of Molecular Structural Biology
Max Planck Institute of Biochemistry
Am Klopferspitz 18
82152 Martinsried
Germany
Email: guoq.tiger at gmail.com
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