[3dem] [External] Re: tilted transfer functions?
Daniel Asarnow
asarnow at msg.ucsf.edu
Wed Mar 2 00:39:55 PST 2022
Hi David,
That's what I thought you meant - as a practical matter, this issue is
resolved by the patch approach. How cryoSPARC averages the patch fit
quality has not been described, but tilted images can be filtered very well
in my experience. The local defocus patch estimates are also generally very
accurate - it's common for CTFFIND to fit a highly tilted image to 6-8Å,
but patch fits of the same image extend to 3-4Å (the programs use the same
definition of the fit resolution), and I've only single-particle estimates
improve on the patch values for large particles like ribosomes or the 20S
proteasome. These notes are all from a single-particle perspective.
Best,
-da
On Tue, Mar 1, 2022 at 10:50 AM Morgan, David Gene <dagmorga at indiana.edu>
wrote:
> Daniel,
>
> Programs such as ctffind give some sort of measure of the quality of the
> CTF estimate by comparing the real data with calculated CTF parameters.
> Unless the programs know how to account for tilt when doing this
> comparision, the quality of the fit will be artificially bad because of
> Thon ring incoherence due to the tilt.
>
> Since one of the first ways to eliminate bad images acquired with
> automated single particle data collection methods is to "filter out" the
> bad images based on things like the CTF results, this above-mentioned
> measure of the quality of the fit needs to be robust to tilting the grid.
>
> I remember that there once was at least one program that could do this,
> but I haven't been able to find it. I have been told (since yesterday)
> that this is implemented in some alpha releases of cisTEM, for example, and
> I thought that the Grigorieff group had released a stand-alone version of
> that software. But I haven't been able to find a recent version. I will
> hunt through my archives...
>
> --
> politics is more difficult than physics.
> A. Einstein
>
> David Gene Morgan
> Electron Microscopy Center
> 047E Simon Hall
> IU Bloomington
> 812 856 1457 (office)
> 812 856 3221 (3200)
> https://urldefense.proofpoint.com/v2/url?u=http-3A__iubemcenter.indiana.edu&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=e9S-KUSpyN0BzfMknMGLGQnTMbdbaOmnWWW4GvtLTjl4NBkkL_Rue3to5JImcRlg&s=ww1SXln4bcroYv1kOrTj4Ul9D5aVVainWdVqpjmNYyg&e=
> ------------------------------
> *From:* Daniel Asarnow <asarnow at msg.ucsf.edu>
> *Sent:* Tuesday, March 1, 2022 2:49 AM
> *To:* Philip Köck <koeck at kth.se>; Benjamin Himes <himes.benjamin at gmail.com
> >
> *Cc:* Morgan, David Gene <dagmorga at indiana.edu>; 3dem at ncmir.ucsd.edu <
> 3dem at ncmir.ucsd.edu>
> *Subject:* Re: [3dem] [External] Re: tilted transfer functions?
>
> Just to clarify, the patch methods don't require particle locations. The
> gCTF method requires particle locations as input but there's no need for
> them to be real particles; it too just estimates the CTF on the patches.
>
> David, what do you mean by the goodness of fit estimates being incorrect?
> In these methods a goodness of fit measure is used to pick the optimal
> defocus values in the patch, and these values do give superior
> reconstruction resolution and appear to accurately define the real tilt
> axis.
>
> Best,
> -da
>
> On Mon, Feb 28, 2022 at 10:51 PM Philip Köck <koeck at kth.se> wrote:
>
> Hi.
>
>
> There are two separate problems here.
>
> The easier one is to simply determine the defocus in various positions of
> the specimen.
>
> If you only want to work with small cut-outs (such as single particles)
> then you just use the normal CTF-correction with the local defocus. Others
> pointed to software that does that.
>
>
> If you want to work with the image as a whole (for example for 2D
> crystals) the problem is more difficult. Essentially you don't have a CTF
> in that case (it's not a transfer function).
>
> Look at the paper by Ansgar Philippsen (Ultramicroscopy 107 (2007)
> 202–212) if that's what you're after. Also check software for 2D
> crystallography, but I don't what is actually implemented.
>
>
> All the best,
>
>
> Philip
> ------------------------------
> *Från:* 3dem <3dem-bounces at ncmir.ucsd.edu> för Morgan, David Gene <
> dagmorga at indiana.edu>
> *Skickat:* den 28 februari 2022 23:50:32
> *Till:* Daniel Asarnow
> *Kopia:* 3dem at ncmir.ucsd.edu
> *Ämne:* Re: [3dem] [External] Re: tilted transfer functions?
>
> Daniel,
>
> The problem is that the tilt causes the higher resolution Thon rings to
> become out-of-phase, and so the standard programs can't give a decent
> estimate of the goodness of-fit. I vaguely remember that someone had a
> program that took the tilt into account a number of years ago, but a quick
> hunt for it didn't turn up anything.
>
> --
> politics is more difficult than physics.
> A. Einstein
>
> David Gene Morgan
> Electron Microscopy Center
> 047E Simon Hall
> IU Bloomington
> 812 856 1457 (office)
> 812 856 3221 (3200)
> https://urldefense.proofpoint.com/v2/url?u=http-3A__iubemcenter.indiana.edu&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=e9S-KUSpyN0BzfMknMGLGQnTMbdbaOmnWWW4GvtLTjl4NBkkL_Rue3to5JImcRlg&s=ww1SXln4bcroYv1kOrTj4Ul9D5aVVainWdVqpjmNYyg&e=
> <https://urldefense.proofpoint.com/v2/url?u=http-3A__iubemcenter.indiana.edu&d=DwQFAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=XfGX1GAOnfj3mLhTnyN8uolpwG3BMtMXZYm0H1sLXZy6LYulpNRf8T8jKuxWTOju&s=qBVmiqzIFHPhsmMv2GVKS3tE0Y3k35YCkiZM5CmZ2cg&e=>
> ------------------------------
> *From:* Daniel Asarnow <asarnow at msg.ucsf.edu>
> *Sent:* Monday, February 28, 2022 5:45 PM
> *To:* Morgan, David Gene <dagmorga at indiana.edu>
> *Cc:* 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> *Subject:* [External] Re: [3dem] tilted transfer functions?
>
> This message was sent from a non-IU address. Please exercise caution when
> clicking links or opening attachments from external sources.
>
> I think any patch-based CTF program, like the one in cryosparc or gCTF's
> local mode (with evenly spaced coordinates instead of real particle
> locations) would work well. With cryoSPARC there's an easy method to plot
> the tilt axis; you can also choose a specific number X and Y divisions for
> the patches.
>
> Best,
> -da
>
> On Mon, Feb 28, 2022 at 2:41 PM Morgan, David Gene <dagmorga at indiana.edu>
> wrote:
>
> Hi,
>
> What's the best way to evaluate the CTF of an image that has a significant
> amount of tilt?
>
> --
> politics is more difficult than physics.
> A. Einstein
>
> David Gene Morgan
> Electron Microscopy Center
> 047E Simon Hall
> IU Bloomington
> 812 856 1457 (office)
> 812 856 3221 (3200)
> https://urldefense.proofpoint.com/v2/url?u=http-3A__iubemcenter.indiana.edu&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=e9S-KUSpyN0BzfMknMGLGQnTMbdbaOmnWWW4GvtLTjl4NBkkL_Rue3to5JImcRlg&s=ww1SXln4bcroYv1kOrTj4Ul9D5aVVainWdVqpjmNYyg&e=
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