High-resolution refinement in cryoSPARC
In this second part of the Workflow Series, we will explore how to run refinement experiments in cryoSPARC. For a walk-through of high resolution refinement, see our post on the T20S proteasome.)
From the result of a single-class ab-initio reconstruction
For details on how to perform a single-class ab-initio reconstruction, see Figure 1, stream A. Figure 2 below provides an overview of a typical refinement experiment workflow, starting with the output of a single-class ab-initio reconstruction. (The typical ab-initio experiment workflow is described in Part I of the Workflow Series.)
A. It is recommended to first try a refinement using cryoSPARC's default parameters. Generally, most refinement parameters do not need to be modified except for a symmetry specification if required, and masking parameters. By default, cryoSPARC automatically generates masks during refinement (called dynamic masking). We recommend performing a default refinement experiment first, and modifying parameters in subsequent refinements.
To perform a refinement, first ensure you have the dataset of interest selected on the Datasets page. Navigate to the Experiments page and find the result structure from the single-class ab-initio reconstruction that you completed previously. Click on the 'Structure' and 'Particle' buttons below the image projection to select the result structure and corresponding particles for the new refinement experiment. You will notice that the 'Selection' box to the right of the page will change to indicate the structure and particles you have selected.
Note: If you do not click on the 'Particles' button below the image projection, cryoSPARC will by default include all of the particles in that dataset. This is not really an issue when refining the result of a single-class ab-initio reconstruction because by default all of the particles belong to that single class, but failing to select particles for refinement following a multi-class ab-initio reconstruction will cause incorrect results. See the "From the results of a multi-class ab-initio reconstruction" section below for details.
Click 'New Experiment' and select 'Refinement' from the list. Click 'Launch' and 'Enqueue' to commence the refinement.
B. It is recommended to explore the results of the refinement performed in A. to determine whether you are satisfied with the refinement result or if you wish you perform another refinement experiment on the same structure and particles.
C. After reviewing the results, you may wish to change the masking parameters. This can be done in one of two ways, as illustrated.
(1) In D1., you can start another refinement experiment by following the steps described in B., but this time, change one or more dynamic masking parameters from the 'Setup' page. You can get to the 'Setup' page by selecting the structure and particles, clicking 'New Experiment', then 'Refinement', and then 'Setup' from the left-hand sidebar. The dynamic masking parameters can be found under the 'Masking' heading and can be expanded by clicking 'Show Advanced Params'.
(2) In D2., you can import a mask that you have generated externally (by hand or with another program). To do so, navigate to the Experiments page and click on 'Import Volume/Mask'. You will be able to upload your file directly through the web browser, removing the need to find the file on your GPU machine. Be sure to set the 'Imported volume is a mask' parameter to true. After uploading, go to the Launch page and click 'Enqueue'. This will cause cryoSPARC to find, plot, and register your imported mask. Next, from the Experiments page, select the original structure and particles and the newly imported mask that you want to use in the refinement experiment.
After changing the masking parameters or importing the mask, proceed to start the refinement as outlined in B., above.
From the results of a multi-class ab-initio reconstruction
Structures identified using heterogeneous ab-initio reconstruction can also be directly refined. Simply select the structure and particles corresponding to a particular class you wish to refine on the Experiments page, optionally select an imported mask, and click 'New Experiment' and then 'Refinement'.
Note: It is also possible and often useful to select sets of particles from multiple classes together, along with a single structure to perform refinement. For example, if multiple ab-initio classes appear to contain the same structure, this is a natural way to merge the particles from those classes when refining.