Tri-snRNP

Dataset

Ensure you have created a location to keep track of CryoSPARC projects:

Title Value
Title
Description Leave empty
Directory
Create Workspace Enable
Workspace Title Leave default

There are a few prepared jobs that we will import into the project. They were exported from a different CryoSPARC instance and transferred onto the local SSD of your EC2 instance.

  1. Navigate to the project directory:
  2. Make a new directory called imports and navigate into it:
  3. Move J109_nonuniform_refine_new
  4. Move J270_new_local_refine
  5. Move J168_volume_tools

Navigate to the workspace you just created, click on the dropdown next to 'New Job', select 'Import Job' and fill out the corresponding absolute path. Do this for all three jobs. You should see three completed jobs in the workspace:

Import Path Job Type Imported Job Title
Non-Uniform Refinement None
Local Refinement None
Volume Tools Imported Volume 167

Open the local refine and mask, show them overlaid, explain we’re trying to lock in particle poses to that region.

  1. Open the volumes in UCSF ChimeraX

Create two Downsample Particles jobs, one for the particles from the non-uniform refinement and one for the particles from the local refinement. Use a box size of 256 pixels and downsample to 128 pixels.

Parameter Value
Fourier crop to box size
Save results in 16-bit Yes
Recenter using aligned shifts Yes

Create two 3D Variability Analysis jobs, one for the particles from the non-uniform refinement and one for the particles from the local refinement. For both jobs, use the mask we imported (Volume Tools)

Parameter Value
Filter resolution (A)
Cache particle images on SSD No

Running Both Jobs in Parallel

Although we have a single GPU available on the EC2 instance, we can run both 3D Variability Analysis jobs in parallel by using the 'Launch on GPU' functionality, which overrides the typical GPU resource requirements.

  1. Queue the first 3D Variability Analysis job as usual (queue button or right-click menu).
  2. Click on the remaining job, ensure the 'builder' mode is active and click 'Queue' from the sidebar.
  3. Select the tab 'Run on specific GPU' and check the single GPU available on the instance.
  4. Click 'Queue Job' to launch the second job immediately.

To visualize the results of the 3D Variability Analysis, create two 3D Variability Display jobs, one for each of the previous jobs.

Parameter Value
Cache particle images on SSD No

Download each component for both results and open them in UCSF ChimeraX.

Connect the volume from the imported Local Refinement job and downsampled particles derived from the Local Refinement job.

Use default parameters

The job should take ~1.5 minutes to complete.

Parameter Value
Filter input volume res. (A)
Mask threshold

The job should take ~10 seconds to complete.

Use default parameters