CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules
5 affiliations
- 1Department of Anatomy and Cell Biology McGill University 3640 Rue University Montreal QC H3A 0C7 Canada
- 2Life Sciences Institute University of Michigan Ann Arbor MI 48109 USA
- 3Faculty of Fundamental Sciences, Hue University of Medicine and Pharmacy Hue University Hue 530000 Vietnam
- 4Department of Biological Chemistry University of Michigan Ann Arbor MI 48109 USA
- 5Centre de Recherche en Biologie Structurale McGill University 3649 Sir William Osler Montreal QC H3G 0B1 Canada
Abstract
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.
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BibTeX
@article{alagha2026csmt,
title = {{CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules}},
author = {Tina Alagha and Asuva Arin and Nicholas Vangos and Helena Goodey-Parfitt and Hai Nguyen Ngo and Nhat Nam Dau and Minh Hoa Nguyen and Thibault Legal and Michael A. Cianfrocco and Khanh Huy Bui},
journal = {Acta Crystallographica Section D: Structural Biology},
year = {2026},
month = oct,
doi = {10.1107/s2059798326010144},
url = {https://doi.org/10.1107/s2059798326010144},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules
AU - Tina Alagha
AU - Asuva Arin
AU - Nicholas Vangos
AU - Helena Goodey-Parfitt
AU - Hai Nguyen Ngo
AU - Nhat Nam Dau
AU - Minh Hoa Nguyen
AU - Thibault Legal
AU - Michael A. Cianfrocco
AU - Khanh Huy Bui
PY - 2026
DA - 2026/10/14
JO - Acta Crystallographica Section D: Structural Biology
DO - 10.1107/s2059798326010144
UR - https://doi.org/10.1107/s2059798326010144
AB - 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.
SN - 2059-7983
ER -