Hi Shintaro,

You are right: per-particle CTF estimation does have room for
overfitting! Therefore, we perform this under strict separation of the
half-sets (the 'gold-standard'). If you don't do that, then you may
indeed get spurious improvements. The current code is safe. Good
indications that things are going well are continuous changes in defocus
over each micrograph, as can be judged from the logfile.pdf. The exact
approach will be published in our upcoming relion-3 paper. That might
need to be carried over the Summer though...

Regarding the autopicking: it is just taking evenly distributed views
from the 3D reference. If you are worried about this, you can always
provide your own 2D references as templates. We haven't seen any
problems with the 3D approach.

HTH,

Sjors



On 08/01/2018 07:56 PM, Shintaro Aibara wrote:
> Hi Sjors,
>
> Hope you're doing well, and thanks for the new release of Relion3! 
>
> I just thought I'd ask a few questions about some of the new features
> mainly surrounding CTF refinement (and to a degree generally
> per-particle CTF estimation).
>
> *Per particle CTF estimation - when is it okay to do it?*
> Since we are going from 3 parameters per micrograph (U,V,Angle) to
> possibly 2x(# of particles) + 1 or 3x(# of particles) parameters where
> each CTF that needs to fit is presumably noisier than that of the
> whole micrograph, do we have anything to keep potential over-fitting
> in check? We seem to be introducing a tonne more fitting parameters
> with less data. 
>
> I seem to get the impression that per-particle CTF/CTF refinement
> always improves the reported resolution by about 0.2 A (for a 2.8-3.5
> A map) and this worries me slightly. It is quite hard to tell whether
> the map has been genuinely improved or not at this level. However, I
> suppose if this is the trend, we must all start doing it to make sure
> our reconstructions keep up with the perceived average resolution of
> the community.
>
> *CTF refinement - any concerns for independent halfsets?*
> I noticed that the way CTF refinement works has been described to be
> comparisons against high resolution reference projections. Presumably
> this would normally be the output of a refinement run, where two
> halfmaps have been combined. Would this mean then that there is
> potential to introduce correlation at high resolution between the
> independent halfsets since the particles have "seen" the combined map?
> As I understand half-sets are also randomized every time you run a
> refinement and so even if CTF refinement was being done against
> half-maps the potential problem for the independent half-sets to be
> not quite independent still remains.
>
> An empirical observation I made was that my FSC of a map after CTF
> [log in to unmask] was very close to the FSC of the refinement without CTF
> refinement @0.143 (equaling about an improvement in 0.2 A or so, as
> above). Perhaps just a coincidence, but was food for thought. 
>
> *3D autopicking - picking particles with views that may not exist?*
> Are there any adverse effects from picking with views of a
> reconstruction that may be severely preferred (usually I find this
> manifests as a streaky mess). It just seems slightly concerning about
> biasing the picking with views that may or may not actually be present
> in the dataset. Are there any decision making within the algorithm to
> decide whether a projection is used as a reference or not, or are all
> projections used?
>
> Best wishes
> Shintaro
>
>
>
> On Wed, Aug 1, 2018 at 6:17 PM, Sjors Scheres
> <[log in to unmask] <mailto:[log in to unmask]>> wrote:
>
>     Dear EM-ers,
>
>     After multiple months of in-house testing at MRC-LMB and the
>     SciLifeLab, and several months of beta-testing by a few external
>     expert groups, we think that RELION-3.0 is now ready for a more
>     general round of beta-testing. To that purpose, you can download
>     it from:
>
>     git clone https://bitbucket.org/scheres/relion-3.0_beta.git
>     <https://bitbucket.org/scheres/relion-3.0_beta.git>
>
>     The attached betaGuide.pdf contains a list of new features, and
>     instructions on how to install and provide us with your feedback.
>     Please read it carefully. As always, please use the ccp-em email
>     list (and not a direct email) to ask questions on how to use
>     RELION. We are grateful for your detailed bug reports through the
>     issue tracker on bitbucket, which will help to make RELION better.
>
>
>     Have fun,
>
>     Jasenko, Takanori, Bjorn, Dari, Erik & Sjors
>
>     --
>     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
>     <http://www2.mrc-lmb.cam.ac.uk/groups/scheres>
>
>
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>
>
>
>
> --
> Yours Sincerely,
> Shintaro Aibara

--
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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