Helical Reconstruction of Amyloids in cryoSPARC

Amyloid-mediated proteotoxicity underlies more than 50 human diseases. Cryo-electron microscopy (cryo-EM) analyses have yielded hundreds of in vitro and patient-derived amyloid structures, establishing direct links between filament morphologies and specific pathological conditions. 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 re-processing and validation of publicly deposited datasets, we demonstrate current capabilities and identify 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 largescale 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 toward addressing fundamental biological questions that enhance our capacity for treatment discovery.