A. Baumannii MLA Complex

Dataset

Learning objectives

Importing results into CryoSPARC, 3D classification techniques. Note: Use the outputs of the previous step as the inputs for the next step, unless otherwise noted.

Title Value
Title
Description Leave empty
Directory
Create Workspace Enable

To import a result group , we'll need to link the directory where the file are located to a location within the CryoSPARC project directory that was just created.

  1. Navigate to the project directory:
  2. Make a new directory called imports and navigate into it:
  3. Create a symbolic link from the source data into the imports directory:

Use the job builder to create an Import Result Group job.

Parameter Value
Absolute path to .csg file

Select all exposures with a maximum CTF Fit Resolution (A) of 6A.

You should have 899 exposures to proceed with.

Parameter Value
Minimum particle diameter (A)
Maximum particle diameter (A)
Use circular blob False
Use elliptical blob True

Select pick locations with an NCC >= 0.23 and Power Score > 80 and < 150.

You should end up with around 84K particle locations.

Parameter Value
Number of CPU cores
Extraction box size (pix)
Fourier crop to box size (pix)

Parameter Value
Number of GPUs to parallelize

Choose a set of 2D classes that look sharp and display secondary structure features.

You should end up with around 36K particles.

Use default parameters

Parameter Value
Symmetry

Parameter Value
Point group symmetry

  1. Download the refined volume from the previous Homogeneous Refinement job.
  2. Open the map in UCSF ChimeraX, and note the opened structures model ID as <model_id> .
  3. Decrease the threshold until you can clearly see both "lobes" (these are the MlaB domains, located opposite from the 6-fold symmetric MlaD domain). Note the threshold value <threshold_number> .
  4. Open the Map Eraser (Under Tools > Volume Data > Map Eraser ).
  5. Erase the micelle and the 6-fold symmetric MlaD domain.
  6. Use to binarize the density map of the MlaB domain, to convert it to a mask.
    • Set all density values outside of the MlaB domain to 0:
    • Set all density values within the MlaB domain to 1:
  7. Save the mask to your Desktop:
  8. Upload the mask to your workstation via scp :

Mask generation in ChimeraX

Parameter Value
Volume data path
Type of volume being imported

Parameter Value
Type of input volume
Type of output volume
Threshold (must set to process mask)
Dilation radius
Soft padding width

Input Source
Particle stacks Particles from Symmetry Expansion
Initial volumes Leave empty
Solvent mask Leave empty
Focus mask Mask from Volume Tools
Parameter Value
Number of classes
O-EM batch size (per class)
O-EM learning rate init
Maximum number of F-EM iters
O-EM learning rate halflife (%)
Output results after every F-EM iteration No

Launch this job in parallel with 3D classification (using particles from symmetry expansion).

Input Source
Particle stacks Particles from Symmetry Expansion
Mask Mask from Volume Tools
Parameter Value
Filter resolution (A)

Parameter Value
Downsample to box size

Parameter Value
Output mode
Downsample to box size

Pick around 90 particles from 6 different exposures (density of ~15 particles per exposure), using the exposures from Manually Curate Exposures.

The Blob Picker Tuner automatically finds optimal blob picker parameters, using user-provided manual picks. You may launch the blob picker tuner using the manual picks from the previous step, together with the exposures from Manually Curate Exposures.

Parameter Value
Particle agreement distance (A)