HA Trimer in CryoSPARC Live
For the second part of this case study we'll re-process the HA Trimer dataset using CryoSPARC Live.
| Title | Value |
|---|---|
| Project | Select the project you created in the previous step (e.g. P1) |
| Title |
In CryoSPARC Live, the Configuration tab houses all the parameters required to set up the session. Click on 'Configuration' in the sidebar to navigate to it.
Compute Resources
| Parameter | Value |
|---|---|
| Preprocessing Lane | Lane default (node) |
| Number of Preprocessing GPU Workers | |
| Reconstruction Lane | Lane default (node) |
| Auxiliary Lane | Lane default (node) |
| Use SSD | Enabled (for both reconstruction and auxiliary) |
Parameters
First, toggle the 'Show advanced' checkbox to reveal the advanced parameters (such as gain reference modifiers).
Microscope/Camera Parameter
| Parameter | Value |
|---|---|
| Flip gain ref & defect file in Y? | Yes |
| Raw pixel size (A) | |
| Accelerating Voltage (kV) | |
| Spherical Aberration (mm) | |
| Total exposure dose (e/A^2) |
Blob Picker
| Parameter | Value |
|---|---|
| Minimum particle diameter (A) | |
| Maximum particle diameter (A) | |
| Use elliptical blob | Yes |
Particle Extraction
| Parameter | Value |
|---|---|
| Extraction box size (pix) |
Click 'Apply to All' to apply the parameters.
Exposure Groups
| Parameter | Value |
|---|---|
| Directory to watch | |
| File name wildcard filter | |
| Gain Reference Path |
Click 'Enable' to enable this exposure group.
At this point your session 'Start Checklist' should be green. Click 'Start Session' at the top of the interface to begin processing.
Use the 'Overview' tab to get a sense of exposure data for this session. You can filter exposures from downstream processing by applying thresholds, similar to Exposure Curation.
Add a threshold for CTF Fit Resolution (A) to only include exposures between 0 and 6A.
The blob picker will automatically pick particles from the exposures. You can monitor the progress of the active picker in the 'Picking' tab and set thresholds similar to the Inspect Picks job.
Once you have a sufficient number of initial picks, you can begin Streaming 2D Classification, which will monitor new exposures that are processed and classify picks into the relevant 2D class.
You can restart this process at any time by clicking on the gear icon and selecting 'Restart'.
Once you're happy with the initial 2D classification, click the gear icon in the '2D Classification' tab and select 'Stop'.
Navigate to the 'Ab-initio' tab and select 'Start'.
Once you have an ab-initio structure loaded in the session you can begin Heterogeneous Refinement.
| Parameter | Value |
|---|---|
| Symmetry |
At any time you can pause the session and choose to start it at a later time. Click the 'Pause Session' button at the top of the interface to pause the session.