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CCPEM  August 2018

CCPEM August 2018

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Subject:

Re: RELION 3.0 Bayesian particle polishing and binning questions

From:

Takanori Nakane <[log in to unmask]>

Reply-To:

Takanori Nakane <[log in to unmask]>

Date:

Wed, 29 Aug 2018 19:42:11 +0100

Content-Type:

text/plain

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Parts/Attachments

text/plain (62 lines)

Hi,

Just to make sure, when you re-ran Polishing, did you do the following?

1. Re-extract particles in the desired box size and pixel size
2. Re-refine them
3. Run polishing

Also, what happens if you simply use the default parameters?

Best regards,

Takanori Nakane

On 2018/08/29 18:06, Mark Herzik wrote:
> Hi All,
>
> First, apologies for the long email.
>
> We have a dataset of a small protein (<150 kDa) collected at a fairly high magnification (0.56 Å/pixel) that we have been trying to process to its fullest using the new Bayesian particle polishing implementation in RELION 3.0. We have hit a proverbial wall in our processing and are hoping some of our fellow EMists could provide some insights as to how best to proceed.
>
> Implementing Bayesian PP with data that have been binned 2x2 (1.12 Å/pixel) using a 384 pixel box size yields robust particle trajectory alignments, nice dose-weighting diagnostics, and yields a 3D reconstruction that is Nyquist limited (2.3 Å resolution) without CTF refinement. Great.
>
> However, we are now trying to extract shiny particles with a smaller downsampling (to lower the Nyquist-limiting frequency) and larger box size (to decrease the effects of CTF delocalization, which is quite severe at 0.56 Å/pixel and 200 kV) without much success.
>
> What we have tried:
> 1) Polishing the unbinned data or data binned 1.5x1.5 using parameters trained against those data results in very noisy particle trajectories, a poor B-factor and dose-weighting scheme, and a worse resolving reconstruction as a result of these efforts (~2.6 Å resolution). Quite surprising given that these steps utilize the same particle stack that resulted in the 2.3 Å resolution reconstruction mentioned above.
>
> 2) Polishing the unbinned data or data binned 1.5x1.5 using parameters trained against the binned 2x2 data yields better results than those obtained in scenario 1 but worse than the binned 2x2 data processing in their entirety (~2.5 Å resolution versus 2.3 Å resolution).
>
> 3) Training the binned 1.5x1.5 data using a small box size (256 pixels, which aids the training step) and then trying to extract the shiny particles with a larger box size (512 pixels) -> RELION crashes with the following error:
>
> ERROR:
> Box size cannot be changed without re-estimating motion - reference pixel size (0.84 A) is not an integral multiple of movie pixel size (0.56 A)!
>
> SO we wonder, is there a protocol currently implemented within RELION 3.0 that can use the particle trajectories from downsampled data (the binned 2x2 data in our case) but then extract the shiny particles using a user-inputted pixel size and box size?
>
> Any and all help will be greatly appreciated.
>
> Thanks,
> -Mark
>
> Laboratory of Dr. Gabriel Lander
> Helen Hay Whitney Foundation Postdoctoral Fellow
> The Scripps Research Institute
> Integrative Structural and Computational Biology
> 10550 N. Torrey Pines Rd. HZ 175L
> La Jolla, CA 92037
> Office: (858) 784-9499
>
>
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