Abstract 1475 High-throughput Collaborative in situ Cryo-ET Enables the Molecular Atlas of C. reinhardtii

In situ Cryo-Electron Tomography (Cryo-ET) has emerged as the method of choice to investigate the structures of biomolecules in their native context. Currently, thicker unicellular and, more recently, multicellular samples are machined using cryo-focused ion beam milling (cryo-FIB) to produce thin sections (lamellae). However, Cryo-FIB milling using conventional dual-beams suffers from recontamination, limited throughput, and manual transfers; compromising both data quality and volume. A next-generation Arctis cryo-plasmaFIB improves both lamellae quality and throughput. Combined with recent advances in data collection and processing, it is now possible to generate (>1000 tilt series) in situ datasets which will facilitate high throughput collaborative in situ structural biology. As a proof of principle, we have generated a large (1800 tilt-series) curated in situ cryo-ET dataset for C. reinhardtii, which offers an established model system to study fundamental cellular processes. During initial studies, we were able to obtain sub-10 Å subtomogram averages of biomolecular complexes ranging in size from a few mega Daltons, for example, 80S ribosomes, to several hundred kilo Daltons, for example, RuBisCo. Leveraging this high quality and the large-scale dataset, we are generating a high-resolution molecular atlas of C. reinhardtii by one of its kind collaborative computing endeavor to identify, average, classify, and curate identifiable macromolecular complexes. Additionally, we are making the minimal TOMOMAN project, which facilitates the ease of further analysis, available to the larger cellular cryo-ET community via EMPIAR. Furthermore, this resource will be browsable along with accompanying organelle and particle level annotations to be continuously enriched by community efforts. We envision that these efforts will result in a pioneering and a growing resource for the cell biology community and beyond.