Yes - they are ~200Å systematically different from those estimated using low res (20-6Å) data
Oli
> On Jun 16, 2017, at 9:41 PM, Reza Khayat <[log in to unmask]> wrote:
>
> Is it possible to estimate the defocus parameters from the ice Thon rings?
>
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
>
> ________________________________________
> From: Collaborative Computational Project in Electron cryo-Microscopy [[log in to unmask]] on behalf of Oliver Clarke [[log in to unmask]]
> Sent: Friday, June 16, 2017 5:27 PM
> To: [log in to unmask]
> Subject: Re: [ccpem] Super-res plus lower mag to improve CTF correction?
>
> Hi Jason,
>
> Because your camera DQE depends on the fraction of camera Nyquist and not the pixel size on specimen, if you turn down the mag you will be getting even less of the high-resolution signal you are looking for.
> Hmm - yes of course in terms of DQE - but what I was actually thinking was that by increasing the total number of particles per micrograph (while keeping the effective pixel size approximately the same, and in a region relative to the expected resolution where the DQE doesn’t much matter), the SNR of the power spectra from the protein contribution would increase. Is this wrongheaded?
>
> I am not familiar with the program you are using to do CTF at the bottom, but if the green line is data and the orange is fit then the discordance between peak locations in the 3-4A regime may indicate an incorrect value for Cs or amplitude contrast.
> It’s CTFFIND4.1.8, operating on movie frames, but I get similar results in other programs - the Cs and AC are both correct. I think it is due to there actually being two effective defocus values - one for the solvent (which gives the Thon rings in the 3-4Å range) and one for the protein (the lower res rings). In fact, I can fit either set of rings very well independently, but there is no choice of parameters that fits both at the same time, and the difference in estimated defocus between the two is ~200Å.
>
> I attribute this as being due to the protein particles clustering near the air water interface, but I’m not 100% sure on that.
>
> Cheers
> Oli
> Good luck,
> Jason Kaelber
> ________________________________
> From: Collaborative Computational Project in Electron cryo-Microscopy <[log in to unmask]<mailto:[log in to unmask]>> on behalf of Oliver Clarke <[log in to unmask]<mailto:[log in to unmask]>>
> Sent: Friday, June 16, 2017 3:41:23 PM
> To: [log in to unmask]<mailto:[log in to unmask]>
> Subject: [ccpem] Super-res plus lower mag to improve CTF correction?
>
> ***CAUTION:*** This email is not from a BCM Source. Only click links or open attachments you know are safe.
> ________________________________
> Hi all,
>
> When collecting RyR data (and I guess likewise for other large particles), the number of particles per image is relatively low, and the Thon rings (from protein, not solvent) are consequently relatively weak (see attached), compared to samples of small proteins where we have 100s of particles per micrograph.
>
> I’m wondering if this may be limiting resolution due to poor CTF correction, and whether going to one mag lower to increase the number of particles per micrograph, but using super res to keep a similar effective pixel size, might be a viable strategy to improve power spectra for better CTF correction.
>
> Does anyone have any experience/thoughts re this approach? Or is it dumb for reasons that aren’t occurring to me on a friday afternoon?
>
> Cheers
> Oli
>
> <gc_mic_00046_ctf_avrot.jpg>
>
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