• cryoSPARC Homepage
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• Start cryoSPARC on your EC2 instance if you haven't already:
  • module load cryosparc
  • cryosparcm start
• You can access cryoSPARC via EC2_INSTANCE_URL:39000
• You can access cryoSPARC Live via EC2_INSTANCE_URL:39006
• Projects can be created in the following directory: /workshop/cryosparc_projects
• Case study data is available in the following directory: /workshop/tutorial_data/cryosparc/
• For each cryoSPARC project, set the project-level parameter for SSD Caching to 'Disabled by default'

Interactive job: accept all exposures below a CTF Fit Resolution (A) of 6.

Interactive job: select picks with an NCC >= 0.2 and Power Score > 824 and < 2955

Interactive job

Use default parameters

Use the same parameters as the previous steps.

Select 'Apply to All' with all parameters set, enable the exposure group and start the session.

Interactive job: select picks with an NCC >= 0.2 and Power Score > 635 and < 1045

Interactive job

Use default parameters

Additional Steps

• Create an import folder in your project directory and symlink the Baum folder into it:
  • cd /workshop/cryosparc_projects/PX (replace Px with your project ID)
  • mkdir imports && cd imports
  • ln -s /workshop/tutorial_data/cryosparc/Baum Baum
• Create a workspace called ‘Processing’
• Import result group with exposures
  • /workshop/cryosparc_projects/PX/imports/Baum/P211_J5_exposures/P211_J5_exposures_exported.csg (replace Px with your project ID)
• Manually curate exposures
  • CTF fit resolution (2.96 → ~6.0) → ~900 exposures
• Manual picks
  • Pick ~20 particles on 4-5 micrographs at a broad range of defocus levels (e.g., 25K, 20K, 15K, 10K)
  • Easiest to start at high defocus to see particles clearly at the outset
• Launch blob tuner picker
  • Connect 80-100 picks from manual picker job
  • Connect all ~900 micrographs
  • Set agreement distance to 100 A (roughly diameter of particle)
• Launch blob picker (baked cake parameters)
  • Type: ellipse only
  • Min diameter: 114.29
  • Max diameter: 210.71
• Inspect picks
  • Use blob picker outputs
  • NCC: 0.23
  • Power: 80 - 150
  • ~75-100K particles
• Extract from Micrographs
  • Extraction box size: 256px
  • Fourier crop: 200px
• Queue 2D Class
• Queue Select 2D Class
  • Select ‘good’ classes (~50K particles)
• Queue Ab-initio
• Queue homo refine, NU refine (symmetry: C2)
  • Run times:
    • NU: 16 min
    • Homo: 4 min
    • Chimera Mask Generation
  • https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/local-refinement/mask-selection-and-generation-in-ucsf-chimera
  • Descrease threshold until you can clearly see both lobes:
    • (Somewhere between 0.13 and 0.16)
  • Use the map eraser tool to remove the micelle and six ‘legs’
  • vop threshold #3 minimum 0.17 set 0
  • vop threshold #4 maximum 0.16 setMaximum 1
  • Save as test_mask.mrc
  • Copy over the mask:
    • scp baum_mask.mrc participant@18.205.234.102:/workshop/cryosparc_projects/P6/imports/Baum/baum_mask.mrc
• Import 3D Volumes
• Volume Tools
  • Mask → Mask
  • Threshold: 0.13 (or whatever you used!), Dilation radius: 5, Soft padding width: 15
  • Download the mask and overlay it on the original volume in Chimera
• 3D Classification (BETA)
  • Use the following parameters:
    • 3 classes
    • 6 A
    • 2 O-EM iterations
    • 10 final iterations
    • 2000 particles / class,
    • Learning rate of 0.75
    • Learning rate half-life of 100
    • Auto-tune initial class similarity off
• Open the series in Chimera
  • open path/to/series/*.mrc vseries true
• 3D Variability
  • Connect particles/mask from homo refine (or try NU refine)
  • Set filter resolution to 6 A
  • ~16 min
• 2 X 3D Variability Display
  • simple mode, intermediates mode
  • for both: downsample to box size 128

To start processing, we will need to create a container for jobs to run. Select the 'New' button from the projects page and enter the following details:

Interactive job

Use default parameters

To start processing, we will need to create a container for jobs to run. Select the 'New' button from the projects page and enter the following details:

• Create import directory and symlink files:
  • cd /workshop/cryosparc_projects/PX
  • mkdir imports && cd imports
  • ln -s /workshop/tutorial_data/cryosparc/ATPase

To start processing, we will need to create a container for jobs to run. Select the 'New' button from the projects page and enter the following details:

• Create import directory and symlink files:
  • cd /workshop/cryosparc_projects/PX
  • mkdir imports && cd imports
  • ln -s /workshop/tutorial_data/cryosparc/ApoF

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