Helical reconstruction of amyloids in cryoSPARC.

Amyloid-mediated proteotoxicity underlies over 50 diseases. Cryo-EM establishes direct links between filament morphologies and pathology, although microbial functional amyloids illustrate that this fold can evolve to serve physiological roles, unlike their pathogenic counterparts. Despite the growing popularity of the processing software cryoSPARC for single-particle analyses, RELION remains the dominant software platform for performing helical reconstruction of amyloid structures, highlighting an area for further development. Here, we present comprehensive processing guidelines for helical reconstruction of helical amyloids using cryoSPARC. Through systematic reprocessing and validation of publicly deposited datasets, we demonstrate the current capabilities and identify the key limitations, emphasizing the need for amyloid-specific parameter optimization within cryoSPARC workflows. Our findings showcase a potential for developing unsupervised processing workflows to meet the demanding throughput requirements of time-resolved in vitro studies and large-scale compound-screening initiatives, thereby accelerating therapeutic drug development. Ultimately, our goal is to shift the focus of amyloid cryo-EM from computationally intensive processing challenges towards addressing fundamental biological questions that enhance our capacity for treatment discovery.