[3dem] [External] Re: Cs question
Sjors Scheres
scheres at mrc-lmb.cam.ac.uk
Tue Feb 17 08:17:04 PST 2026
Hi Mike,
Yes, exactly! This is why one can do postprocessing in relion with a new
pixel size if one finds out later on in the project that the original
pixel size was incorrect: so no need to reprocess the entire data set.
HTH,
Sjors
On 17/02/2026 16:14, Schmid, Michael F. wrote:
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> --
>
> Great thread!
> I might add that incorrect pixel size can bite you later on. If your
> model seems squished into the map, or wonky Ramachandran angles
> appear, ESPECIALLY for large structures spanning hundreds of Å, you
> may have pixel size problems. The refinement programs will always try
> to do their best, and for small structures, the coordinates can slop
> their way into a map, but having the correct pixel size is optimal.
> (I agree that TMV is a very good ruler, Ruben. It shows your Brandeis
> training!).
> Mike
>
> *From: *3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Sjors Scheres
> via 3dem <3dem at ncmir.ucsd.edu>
> *Date: *Tuesday, February 17, 2026 at 12:43 AM
> *To: *3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> *Subject: *Re: [3dem] [External] Re: Cs question
>
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> Dear EM-ers,
>
> Just to add to Ruben's and Takanori's comments: errors in the assumed
> pixel size are relatively common. They are therefore a likely source of
> the odd Cs values reported by others in this thread. Also, there is no
> real need to run whole refinements with artificially incorrect values to
> see what happens, as one can calculate exact CTF curves and examine at
> what frequency large (e.g. 90-deg) phase differences start to appear.
> Takanori wrote a visual CTF simulator here:
> https://urldefense.com/v3/__https://3dem.github.io/relion/ctf.html__;!!Mih3wA!EXWf9uWAstQO8sLZGG2EGfQYE84kt8x7vnG0gnOlxVCaC4lUJ9ed-8W5pcZQxXjufBJ7bThoObSIMqHtmruMZzKb1tQ$
>
> Hope that helps,
>
> Sjors
>
>
> On 16/02/2026 23:39, tnakane.protein--- via 3dem wrote:
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> > --
> >
> > Hi,
> >
> > If your pixel size is off, both defocus and Cs are refined to absorb
> the pixel size error.
> > This gives a very good CTF fit but the resulting values are
> non-physical.
> > Please see this:
> >
> https://urldefense.com/v3/__https://relion.readthedocs.io/en/release-3.1/Reference/PixelSizeIssues.html*cs-and-the-error-in-the-pixel-size__;Iw!!Mih3wA!FsTUSEahBrII222EDKqoF8yl3yDTXuEv4acHbTZs6iNxcUeVcnEIrQvLLVuClPKv_GJTEcTuc9W8UftXyAmk3V7XgCWFz-hcvPg$
> >
> > Best regards,
> >
> > Takanori Nakane
> >
> > ________________________________________
> > From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Morgan, David
> Gene via 3dem <3dem at ncmir.ucsd.edu>
> > Sent: 17 February 2026 8:31
> > To: zbyszek; Ruben Diaz Avalos
> > Cc: 3dem at ncmir.ucsd.edu
> > Subject: Re: [3dem] [External] Re: Cs question
> >
> > Hi,
> >
> > Thanks for that information. We have come up with a Cs value on a
> Thermo Arctica that is significantly different from what Thermo says
> it should be (our value is about 3.0 while Thermo says it should be
> 2.7) and so I wondered what others have found.
> >
> > I was surprised that our refined value was so different (more than
> 10%). However, I just looked at changes in calculated CTF curves for
> these very different values of Cs, and the changes are tiny. Probably
> too small to have any impact on the structure...
> >
> > I would still like to know whether others find refined values near
> manufacturers' suggestions, and whether our value is an outlier.
> >
> > Again, thanks for the information.
> >
> > --
> >
> > NOTE: my email has changed to dagmorga at iu.edu.
> > Email to dagmorga at indiana.edu will stop being
> > forwarded later this year.
> >
> > politics is more difficult than physics.
> > A. Einstein
> >
> > David Gene Morgan
> > Electron Microscopy Center
> > 043E Simon Hall
> > IU Bloomington
> > 812 856 1457 (office)
> > 812 856 3221 (3200)
> >
> https://urldefense.com/v3/__http://iubemcenter.indiana.edu__;!!Mih3wA!FsTUSEahBrII222EDKqoF8yl3yDTXuEv4acHbTZs6iNxcUeVcnEIrQvLLVuClPKv_GJTEcTuc9W8UftXyAmk3V7XgCWFVFWjLz8$ <https://urldefense.com/v3/__http://iubemcenter.indiana.edu__;!!Mih3wA!BYhqQMW_gvAIW4-KTWwsQmFf1v0aA1iUQyvoIoixPEz59Vol28xVqHV8QXDqWPJpa6YvgWiNdFLohNrw3hk$>
> > ________________________________
> > From: zbyszek <zbyszek at work.swmed.edu>
> > Sent: Monday, February 16, 2026 6:18 PM
> > To: Ruben Diaz Avalos <rdiaz at lji.org>
> > Cc: Morgan, David Gene <dagmorga at iu.edu>; 3dem at ncmir.ucsd.edu
> <3dem at ncmir.ucsd.edu>
> > Subject: [External] Re: [3dem] Cs question
> >
> > You don't often get email from zbyszek at work.swmed.edu. Learn why
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> >
> > Hi Ruben and David,
> >
> > I ran similar tests (refinement of Cs) on both Thermo and JEOL
> instruments. On the Thermo systems, I consistently obtained values
> close to 2.7 across at least 10 different instruments for
> high-resolution structures, with small deviations that are well
> explained by Ruben’s description. I also didn’t see any resolution
> improvement from refining the Cs value.
> >
> > Best regards,
> >
> > Zbyszek Otwinowski
> >
> >
> > On 2026-02-16 15:47, Ruben Diaz Avalos via 3dem wrote:
> >
> > Hi David,
> >
> > Nominal Cs is fundamentally a hardware property of the objective
> lens and pole piece geometry, and manufacturers determine it from the
> electromagnetic lens design. For a Krios, the stated value (~2.7 mm)
> is not empirical tuning but a well-characterized design parameter. In
> principle, it should not vary significantly between microscopes of the
> same configuration.
> >
> > What we refine in image processing, however, is not a direct
> physical measurement of the lens geometry, but the coefficient of the
> k^4 term in the CTF phase function. In Zernike language, this
> corresponds primarily to the radially symmetric fourth-order component
> (the Z4^0 term of the wave aberration). In practice, that fitted
> coefficient can absorb small modeling imperfections — residual coma,
> higher-order aberrations not explicitly modeled, envelope
> inaccuracies, or subtle systematic phase errors. So the "refined Cs"
> should be interpreted as the best-fit fourth-order phase term under
> the assumptions of the refinement model.
> >
> > In one of our datasets (TMV, refined to 1.8 Å), the refined Cs
> converged to 2.7 mm when processing the full dataset. Importantly,
> pixel size is extremely well calibrated in this case because TMV
> provides a very precise internal ruler via its layer-line spacing and
> helical repeat, so I am confident that pixel size error is not
> significantly contributing to the k^4 term.
> >
> > Out of curiosity, I split the dataset into three arbitrary subsets
> and refined them independently. The refined Cs values were 2.75 mm,
> 2.69 mm, and 2.8 mm. When I recombined all particles, the final
> reconstruction was essentially identical to the original 1.8 Å map,
> with no meaningful change in resolution or map features.
> >
> > So empirically, at least in this regime, a ~2–3% variation in the
> refined Cs did not have a noticeable impact on the outcome. That is
> not entirely surprising, since the phase error introduced by a small
> fractional change in Cs remains modest over the spatial frequency
> range that carries usable signal, especially compared to the dominant
> defocus term.
> >
> > It would certainly be interesting to test the robustness more
> aggressively by starting from a deliberately incorrect Cs (for example
> 2.5 mm) and seeing whether refinement converges back to the nominal
> value and whether map quality degrades in the process. But based on
> this dataset, small deviations from the manufacturer value appear to
> be well tolerated at ~1.8 Å.
> >
> > Ruben.
> >
> > Ruben Diaz Avalos,
> >
> > La Jolla Institute for Immunology.
> >
> >
> > On Mon, Feb 16, 2026 at 1:07 PM Morgan, David Gene via 3dem
> <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>> wrote:
> >
> > Hi,
> >
> > For those of you who have used any of the image processing programs
> to refine the Cs value for your microscope, how does the refined value
> compare to what the manufacturers claim?
> >
> > As I understand it, the manufacturers simply calculate a Cs value
> for each microscope/pole piece model. I guess I have always assumed
> that those values are relatively accurate, but I don't really have any
> data to support that. Nor am I certain what would constitute
> "relatively accurate."
> >
> > Any thoughts would be appreciated. Thanks.
> >
> > --
> >
> > NOTE: my email has changed to
> dagmorga at iu.edu<mailto:dagmorga at iu.edu <mailto:dagmorga at iu.edu>>.
> > Email to
> dagmorga at indiana.edu<mailto:dagmorga at indiana.edu> will stop being
> > forwarded later this year.
> >
> > politics is more difficult than physics.
> > A. Einstein
> >
> > David Gene Morgan
> > Electron Microscopy Center
> > 043E Simon Hall
> > IU Bloomington
> > 812 856 1457 (office)
> > 812 856 3221 (3200)
> >
> https://urldefense.com/v3/__http://iubemcenter.indiana.edu__;!!Mih3wA!FsTUSEahBrII222EDKqoF8yl3yDTXuEv4acHbTZs6iNxcUeVcnEIrQvLLVuClPKv_GJTEcTuc9W8UftXyAmk3V7XgCWFVFWjLz8$ <https://urldefense.com/v3/__http://iubemcenter.indiana.edu__;!!Mih3wA!GtPRGTEs6TwC_HvS-grWkxuNLZZrTPkNnxibfaqE1R2wuugqzQRLCXxNTJ6tcwkHZ9lKP0Ewa1fN0w9zI0Y$>
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> > --
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> >
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