/ cryosparc tips

cryoSPARC Workflow Series: Part III Sub-classification


Part III of the Workflow Series explores how to work with multiple structures obtained in cryoSPARC following a heterogeneous (i.e., multi-class) ab-initio experiment. (For a walk-through of ab-initio reconstruction, please see Part I of the Series.)

When to use this workflow: You have completed an ab-initio reconstruction with Number of Classes > 1. You have multiple structures and you are interested in further exploring one or more of those structures to see if there may be sub-classes, pieces or conformations present in the data, or you wish to clean up the data to exclude particles that do not reflect a complete structure.

Figure 3 below provides an example of how to perform sub-classification in cryoSPARC, starting with the outputs obtained from a 5-class ab-initio reconstruction. (To enlarge the image, you can open the image in a new tab or download it.)


A. In this example, we noticed that the largest fraction of the particles in the data set belonged to the second structure. To explore whether there were any sub-classes, we selected a number of sub-classes to explore - 3. Correspondingly, we selected the 'Structure' button below the second structure three times, illustrated by the "x 3". We then selected the 'Particles' that were classified by cryoSPARC as belonging to that structure. This setup essentially asks cryoSPARC to sub-classify the selected particles into three separate classes, with each class instantiated using the structure that was chosen.

To commence the sub-classification, click on 'New Experiment' and then select 'Ab-initio'. Click 'Launch' and 'Enqueue' to start the experiment. (Note: The term 'Ab-Initio' in this context is somewhat of a misnomer. Since the classes are instantiated using the selected structure, the reconstruction algorithm will explore starting from that structure in each sub-class. In other words, this experiment is similar to standard reference-based 3D classification.)

B. After reviewing the results of your sub-classification, you may decide to perform additional sub-classifications using the newly generated classes or the original classes.

Note: If you select particles from multiple classes, cryoSPARC will take the union of those particles, i.e., only one copy of each relevant particle will be used, and those particles will be re-classified amongst the number of classes you requested.