EMBO 2025 - CryoSPARC

An online version of this handout is available at embo2025.cryosparc.com

Click a job's UID in the inputs panel to scroll to that job

J5: Micrograph Denoiser (Prerun)

Inputs

No inputs

Custom Parameters

All default parameters

J6: Curate Exposures

Inputs

J5 exposures

Custom Parameters

All default parameters

J7: Blob Picker

Inputs

J6 exposures accepted

Custom Parameters

Minimum Search Diameter (A) 200
Maximum Search Diameter (A) 320
Use denoised micrographs
Min. separation dist (diameters) 0.5

J8: Junk Detector

Inputs

J5 exposures
J7 particles

Custom Parameters

All default parameters

J9: Inspect Picks

Inputs

J8 particles accepted
J8 exposures

Custom Parameters

Calibrate Pick score to CTF
Calibrate Power score to CTF
Auto Cluster
Target power score 200

J10: Extract from Micrographs (Blob Picks)

Inputs

J9 micrographs
J9 particles

Custom Parameters

Number of CPU cores 8
Extraction box size (pix) 450
Fourier crop to box size (pix) 64
Save results in 16-bit floating point

J11: 2D Classification (Generate Templates)

Inputs

J10 particles

Custom Parameters

Number of 2D classes 40
Initial classification uncertainty factor 0.5
Maximum resolution (A) 15
2D zeropad factor 1

J12: Select 2D (Template Images)

Inputs

J11 particles
J11 class averages

Custom Parameters

All default parameters

J13: Template Picker

Inputs

J12 class averages
J8 exposures

Custom Parameters

Use CTFs to filter the templates
Use denoised micrographs
Minimum Search Diameter (A) 300
Min. separation dist (diameters) 0.6

J14: Junk Detector

Inputs

J13 micrographs
J13 particles

Custom Parameters

All default parameters

J15: Inspect Picks

Inputs

J14 particles accepted
J14 exposures

Custom Parameters

Calibrate Pick score to CTF
Calibrate Power score to CTF

J16: Extract from Micrographs (Template Picks)

Inputs

J15 micrographs
J15 particles

Custom Parameters

Number of CPU cores 8
Extraction box size (pix) 450
Fourier crop to box size (pix) 64
Save results in 16-bit floating point

J17: 2D Classification (pre-3D)

Inputs

J16 particles

Custom Parameters

Number of 2D classes 80
Maximum resolution (A) 15
Initial classification uncertainty factor 0.5
2D zeropad factor 1

J18: Select 2D (pre-3D)

Inputs

J17 particles
J17 class averages

Custom Parameters

All default parameters

J19: Ab Initio Reconstruction (bad volumes)

Inputs

J18 particles excluded

Custom Parameters

Number of Ab-Initio classes 3
Number of initial iterations 20
Number of final iterations 30

J20: Ab Initio Reconstruction (good volume)

Inputs

J18 particles selected

Custom Parameters

All default parameters

J21: Heterogeneous Refinement

Inputs

J16 particles
J20 volume class 0
J19 volume class 0
J19 volume class 1
J19 volume class 2

Custom Parameters

Refinement box size (Voxels) 64

J22: Extract from Micrographs (3D Curated)

Inputs

J16 micrographs
J21 particles class 0

Custom Parameters

Extraction box size (pix) 450
Number of CPU cores 8
Fourier crop to box size (pix) 300
Save results in 16-bit floating point

J23: Volume Tools (flip hand)

Inputs

J21 volume class 0

Custom Parameters

Flip hand

J24: Non-Uniform Refinement (consensus)

Inputs

J22 particles
J23 volume

Custom Parameters

Minimize over per-particle scale
Dynamic mask start resolution (A) 1

J25: Volume Tools (lowpass)

Inputs

J24 volume

Custom Parameters

Lowpass filter (A) 15

J26: Volume Tools (threshold)

Inputs

J25 volume

Custom Parameters

Type of output volume mask
Threshold (must set to dilate, pad, fill holes, or z-clip) {dataset dependent}
Dilation radius (pix) 4

J27: 3D Variability Analysis

Inputs

J24 particles
J26 mask

Custom Parameters

Number of modes to solve 1
Filter resolution (A) 12

J28: 3D Variability Display (simple)

Inputs

J27 particles
J27 volume

Custom Parameters

Downsample to box size 128
Crop to size (after downsample) 100

J29: 3D Variability Display (cluster)

Inputs

J27 particles
J27 volume

Custom Parameters

Downsample to box size 128
Crop to size (after downsample) 100
Output mode cluster
Number of frames/clusters 2

Opening 3DVA volume series

  1. Download both jobs' "All volumes" series outputs.
  2. Unzip them
  3. Rename the directories to "simple" and "cluster"
  4. Run the commands below 

open ~/Downloads/simple/*.mrc vseries true
open ~/Downloads/cluster/*.mrc vseries true

J30: 3D Classification

Inputs

J24 particles
J26 mask

Custom Parameters

Number of classes 2
Filter resolution (Å) 10

J31: Non-Uniform Refinement (class 0)

Inputs

J30 particles class 0
J30 volume class 0

Custom Parameters

Minimize over per-particle scale
Dynamic mask start resolution (A) 1

J32: Non-Uniform Refinement (class 1)

Inputs

J30 particles class 1
J30 volume class 1

Custom Parameters

Minimize over per-particle scale
Dynamic mask start resolution (A) 1

Comparing with an atomic model

  1. Open the active state model by running
    open 8OLT
  2. Use the "translate selected models" right mouse mode, either by clicking it in the GUI or running
    ui mousemode right 'translate selected models'
  3. Select the model and move it into the appropriate position
  4. Fit the model into the active map using
    fitmap #{modelID} inMap #{activeID}
    where each ID is replaced with the appropriate number