Bin micrographs

Cryo-EM Data Processing
10

I think you are getting confused by the slider in manual picker - this only affects visualization.

You can extract the particles in whatever box size you want, using the extraction job.

1 Like
11

That is what I was trying to clarify. Thanks

12

I have figured out the issue and it is solved. However I now ran into another problem-

After extracting the particles (> 1 million) and starting 2D classification- I encounter “cache full …”. I tried to empty space on the cache (1TB SSD) without much success. Is there a way to carry out the job (without adding another SSD).

I split the whole data set (extracted particles in 6 groups (in different folders) and tried to load one set at a time for the 2d classification but it failed.

Let me what can be a good solution.

Best Andy

13

Hi Andy,

1M particles, if sufficiently binned, should fit into 1TB scratch just fine - I would recommend binning to 4Å/pix initially, or 8Å/pix if your stack is still too big to fit on the SSD.

Cheers
Oli

14

Hi Oli,

I am placing my next set of questions here that remain unanswered for more than a week, if you can respond to these.

1.While the class averages are well centered, a major population of the auto picked is completely off centered. How can we center the particles in cryo SPARC those going in the reconstruction process .

  1. I binned the autoboxed particles to carry out the 2D class averaging. After the selection of particles from the correct class averages- How can I use the un-binned forms of the selected particles for reconstructions.

  2. how can we set up finer angular steps and shifts in orientation search and refine processes in the reconstruction process.

  3. In the reconstruction process (refine) how can we regulate the
    particle selection based on Figure of Merit or any other criterion to make selection of the best particles from the selected band, suitable for the highest resolution of the density map.

Thanks
Andy

15

Hi Andy,

  1. If the particles are off centered, but the 2D classes are well-centered, re-extracting with recentering (which is on by default) should recenter them.

  2. You can do this by replacing the particles blob in the low level results with the one from the “full scale” particles - see (Initial resolution improvement, then deterioration in Non-Uniform refinement - #9 by wtempel) for an explanation of the process in another context.

  3. In global refinement, this is handled automatically, and I do not believe there is a way to “force” finer angular samplings. In local refinement there is an option to choose the maximum angular sampling (by default 0.2deg).

  4. There is no way to select based particles based on figure of merit (I guess you are thinking like Pmax in relion) in cryoSPARC, to my knowledge. You can select particles based on probability of belonging to a particular class, after 2D or 3D classification, using the Class Probability Filter job type.

Hope that helps

Cheers
Oli

16

Hi Oli,

Few more questions,

  1. How do I introduce an already computed moderate resolution density map as a starting model in the workflow of cryosparc.

I have the particles boxed out, class averaged, classes selected and particles extracted for reconstruction. At this point instead of using Ab-Initio reconstruction I want to use the pre-computed map as the starting mode and continue with the refinement.

How do I get the program (cryosparc) to read this precomputed map into its pipeline.

  1. How can I get the extracted particles (in mrc format)

Thanks.

Andy

17

  1. Import 3D Volumes

  2. Not sure what you mean - are you asking how to export to read into relion? Or another program? It will depend on what you want to do with them (in terms of how best to export)

18

Hi Oli,

Thanks for the 1st reply

for point no. 2, yes, - how to read the particles in another program - like say BSOFT or EMAN2 etc. I want to do some pre-processing for starting the model refinement or generate a starting model using BSOFT or EMAN 2. Can I get the particle images (stacks) from each micrograph (after frame alignment and dose correction).

Also can I get particles ctf corrected before the reconstruction process ?

19

Particle stacks will be located in the extract sub directory of the extraction job.

No, I believe particles are CTF corrected on the fly, so there are no stacks of CTF corrected particles.

20

HI Oli,

Continuing the thread here-

While carrying out class averaging for particles I encountered " illegal memory access was encountered". I do not find a solution to overcome this. Additionally this happened when I am trying to add another set of data to the existing one (which already provided a 3.4 Å resolution map).

Any feedback and a fix to the problem will be helpful.

Best
Andy

21

I’m not sure, but perhaps one of the cryosparc @team can help!

22

@Andy Please can you add more details about the error:

  • cryoSPARC job type
  • where in the logs or interface the error was encountered, with additional lines of output for context
  • the current “physical” box size of the particle stacks and any job parameters that affect the job’s memory complexity (resolution, number of classes, particles, etc.)
  • hardware and software:
  • how the other dataset was added to the existing one, and how the datasets differ
23

A post was split to a new topic: No particles from filament tracer

24

A post was merged into an existing topic: No particles from filament tracer

25

Hi Oli and wtempel,

The above issue also taken care of at present (BUT I will revisit it later to provide you more details). I have another problem.

  1. I recently completely a helical reconstruction with resolution up to 3.6Å. Now I want to generate the whole 3.6Å map to a much larger length (it is a phage tail density map). I will need the asymmetric map (at 3.6Å) so that I can symmetrize it to generate a 1200Å long map (the map is ~256Å long). How to obtain the asymmetric map ?

Is there any alternate way to do it ?

  1. I want to sort out heterogeneity based on the Pitch of the helix. I can generate maps for classifications from the asymmetric map, requested above. Following that How to I carry out the classification using the maps in cryosparc ?

Thanks

Andy

How do I do that?

26

Hi @Andy,
Thank you for posting these interesting questions.

To facilitate use and management of this forum, please post questions 1. and 2. separately as two new topics in the Helical Processing category.

27

Hi Andy,

Can I know how to bin micrographs in the CS? What’s the job name?

28

Hi

I binned the images in BSOFT (or you can do it in any package like EMAN2) and then imported them in Cryosparc.

However if memory serves well, there should be an option in cryosparc, I can possibly reply you back tomorrow or day after tomorrow. Some one from cryosparc team can definitely reply you, sooner but you can try the ‘Fourier crop to box size’.

Best

29

Depending on your stage of the workflow, “binning” can be accomplished in various job types.
For example, see this discussion about Patch Motion Correction.
Additionally, there are the Extract From Micrographs and Downsample Particles job types that can achieve a binning effect.