Hi Han,
You have a good microscope and really good sample!
If you take a look at these entries into the EMDB, you are matching the best published Cryo-EM results!
http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-7770 (1.9Å, 150K particles, with PETG)
http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8908 (2.2Å, 106K particles)
So far all structures with better resolution than 1.9Å for this sample are from crystallography.
So stop worrying and go have fun with the Titan!
Jeppe
-----Original Message-----
From: Collaborative Computational Project in Electron cryo-Microscopy [mailto:[log in to unmask]] On Behalf Of Takanori Nakane
Sent: 15. december 2018 21:20
To: [log in to unmask]
Subject: Re: [ccpem] Requirements to achieve high resolution using less than 20K particles?
Hi,
> By contrast, when I processed RELION tutorial data, merely ~9K
particles from 25 mics was so enough to achieve its Nyquist resolution
(really strong data, I think).
No, the pixel size of the tutorial dataset is 0.885 A.
Its 2.9 A is far from Nyquist.
I think getting 2.17 A from 46 K particles of beta-galactosidase is
good enough.
Best regards,
Takanori Nakane
On 2018/12/15 15:02, 류범한 wrote:
> Hello, CCPEM.
>
> Recently I’ve collected movies of E.coli K12 β-galactosidase, which is regarded as a ‘standard' sample, to examine the full performance of our Titan Krios.
>
> I collected total 370 movies using Falcon3 counting mode at Mag. 97,000 (corresponding to 0.863 Å/pixel).
> And 46K particles from sorted 256 micrographs were processed using Relion 3.0.
>
> For the micrographs, 60% were below 15,000 Å defocus, 77% below 500 Å, and 72% below 5Å max. resolution fit after CTF estimation (mostly 2 or 3 Å fit). The entire micrographs were sorted out to make the data pool very similar with RELION3.0 tutorial dataset (which is also of β-gal).
>
> Then, carefully selected 46K particles from the sorted 256 micrographs were CTF-refined and bayesian polished.
>
> In the end, the final resolution of the reconstructed 3D map from the data was at 2.17 Å after post-processing.
>
> By contrast, when I processed RELION tutorial data, merely ~9K particles from 25 mics was so enough to achieve its Nyquist resolution (really strong data, I think).
>
>
> At this point, I am wondering why the data I obtained could not produce a 3D map at its Nyquist resolution (~1.73 Å), or what are additionally needed for it.
>
> I guess one or a few of the followings I listed can be related to the limitation of my dataset in resolution.
>
>
> ######################################
> 1) Absence of PETG(inhibitor) in the sample — led to higher b-factor?
>
> 2) Use of 70µm condenser aperture other than 50µm (our 50µm one has been contaminated…) —led to (a little) less coherent beam?
>
> 3) Use of 100µm objective lens aperture — I should have avoided to use any aperture for 1.XÅ resolution?
>
> 4) Simply, insufficient amount of data (370movies)— then why RELION tutorial dataset can make it with less than 10K particles? Somehow, Reslog analysis showed a unsaturated curve.
>
> 5) Difference in detector? — The tutorial dataset was recorded using K2, and my movies using Falcon3. But you know Rado have already made 1.6Å apoferritin map using Falcon3..
>
> 6) Absence of energy filter — we just don’t have it.. :(
>
> 7) 300kV ? — Relion tutorial data was recorded under 200kV. So, 200kV may be a little bit advantageous in mean free path and lead to better SNR? But as far as I know, we can of course achieve 1.X Å resolution under 300kV, right?
>
> 8) Need to increase dose per frame a little bit higher? (from 1.0 e/Å2 frame to 1.2 or 1.3?)
> ######################################
>
>
> Considering above, please let me know how I can make better movies and/or results.
>
> My goal is to make a strong data of ~50 movies containing ~20k particles that can achieve 1.X Å resolution.
>
> Any suggestions or advices are welcome.
>
>
> Best regards,
> Han
>
>
> Bum Han Ryu
>
> Postdoctoral Researcher
> Korea Basic Science Institute (KBSI)
> 161 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si
> Chungcheongbuk-do, Republic of Korea
>
> Office) +82-43-240-5329
> Mobile) +82-10-7119-0505
>
>
>
>
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