Jasenko is not a member of ccpem and cannot post here,
so I am forwarding this to the list instead.
Takanori
On 2018/08/29 22:34, Jasenko Zivanov wrote:
>
> Hi Mark,
>
> If you were to use the trajectories computed from the 2x2-binned images,
> the program would allow you to extract boxes of arbitrary size. The
> result would still be at 1.12 Å/pixel, though, and you are already
> hitting Nyquist there.
>
> The reason we don't currently support changing the box size for
> non-integral pixel-size ratios is that the box size also has to be a
> natural number in movie pixels. For a given box size in
> reference-pixels, a larger box has to be extracted from the movie, and
> then Fourier-cropped to the specified target size. If the size of the
> larger box is not an integral multiple of the size of the smaller box,
> then one could end up having to extract a box of fractional size, which
> isn't possible without resampling.
>
> In your specific case, the numbers would accidentally work out, though:
> In order to obtain a 512-pixel box at 0.84 Å/pixel, you would have to
> extract a 768-pixel box from the 0.56 Å/pixel movie - which is indeed a
> natural number.
> So, if you like, you could comment out lines 45-51 in
> src/jaz/motion/frame_recombiner.cpp to deactivate this condition, and
> use motion_refine with --comb_box 512.
>
> Alternatively, you could also just pad your reference maps (and the
> mask) to 512 pixels with relion_image_handler (don't forget the --o
> parameter or it will overwrite the input image!), and run motion refine
> with those. This would be probably simpler, but it might produce less
> reliable trajectories (see next sentence).
>
> A word of warning, though: although a larger box does contain more of
> the delocalized higher frequencies, its signal-to-noise ratio is worse
> for the lower frequencies due to the empty space around the molecule.
> This makes both motion estimation and the refinement itself less reliable.
>
> We had the same problem with a tiny protein, gamma-secretase. If the box
> is large enough to contain all of the higher frequencies, then it is
> also mostly empty. In the end, we obtained better results with a box of
> insufficient size - and that dataset was collected on a 300 keV
> microscope, where the delocalization effect is far less severe.
>
> Hope this helps.
>
>
> Best regards,
> Jasenko
>
>
>
>
> On 08/29/2018 07:42 PM, Takanori Nakane wrote:
>> 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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>>>
>>
>
>
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