CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules.

Microtubules are cytoskeletal filaments that are involved in intracellular transport, cell division and motility. Despite their biological importance, determining their high-resolution structures via cryo-electron microscopy remains a significant technical challenge due to their polymorphism and pseudo-helical assembly. Current processing workflows are complex, often requiring the integration of multiple software packages and custom scripts, which creates a steep learning curve for many research groups. To address these limitations, we introduce CsMT, a streamlined workflow implemented entirely within the CryoSPARC environment and using synthetic references. CsMT simplifies microtubule reconstruction by utilizing a novel protofilament-pair classification approach, which effectively handles the inherent pseudo-symmetry and structural heterogeneity of microtubules with minimal manual intervention. Our workflow is versatile, and is capable of processing both undecorated and decorated microtubules while accurately determining seams and performing high-resolution refinement. We demonstrate the efficacy of this workflow by achieving 2.3 and 2.7 Å resolution reconstructions of homotypic and heterotypic maps of undecorated microtubules, matching the best-resolved microtubule structures in the field. By unifying the pipeline into a single and portable workflow, CsMT enhances reproducibility and accessibility, empowering more laboratories to explore the structural biology of microtubules and associated proteins, yielding new insights into their function.