CRYO-EM STUDIES OF SACCHAROMYCES CEREVISIAE SEPTIN COMPLEXES: A COMPLETE COLLECTION OF INTERFACES

Septins are cytoskeletal GTP-binding proteins essential for cell division, polarity, and membrane remodeling. Yeast septins are particularly relevant as they were first discovered in Saccharomyces cerevisiae. Although structures of individual septin subunits and smaller complexes have been solved independently, a structure of the entire yeast septin octamer showing all subunits interacting with their respective partners simultaneously remains unresolved. To bridge this gap, the four canonical S. cerevisiae septins (Cdc11, Cdc12, Cdc3, Cdc10) were heterologously expressed in E. coli and purified, with their oligomeric state confirmed by SEC-MALS. Two samples were produced for cryo-EM grid preparation: an octamer containing all four subunits and a Cdc11-Cdc12 tetramer, the latter purified under low-salt conditions to enable formation of the polymerization interface. Cryo-EM data were collected on a Titan Krios equipped with a Falcon 4i camera and a Selectris energy filter at IGBMC, France. The initial octamer dataset suffered from preferred orientation, impairing map reconstruction; furthermore, on-grid polymerization yielded heterogeneous particle sizes that complicated processing. Consequently, a second construct containing a truncated version of Cdc11 was prepared to prevent polymerization, and data was collected using grid tilting to mitigate preferred orientation. Both datasets were merged to maximize the particle count during processing, yielding superior 2D classes and a preliminary map with reduced preferred orientation, which is currently being refined. Additionally, the Cdc11-Cdc12 sample dataset was also collected using tilting, and a preliminary tetramer map is also being processed. Ultimately, the resolution of both structures will allow us to obtain a complete landscape of septin interfaces in a context closer to their natural state, avoiding the artifacts associated with crystal packing and crystallization environments. Furthermore, having structural data on all interfaces will allow us to understand the differences in their in vitro stability, explaining, for example, why the polymerization interface is sensitive to salt while the others are not. This work was supported by CAPES (88887.641936/2021-00) and FAPESP (2020/02897-1 and 2022/00125-7).