Related Experiment Video
Updated: Apr 29, 2026
A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion
Published on: January 31, 2022
A cryo-EM processing pipeline for microtubules using CryoSPARC
Daniel Zhang1, Hugo Muñoz-Hernández1, Pavel Filipcik2
1Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland.
Abstract:
Microtubules are cytoskeletal filaments that are typically characterized by a discontinuous helical lattice of α/β-tubulin heterodimers. Microtubules can also adopt variable lattice architectures both in vitro and in cellular contexts. Pseudo-helical averaging processing strategies have been developed to generate cryo-EM reconstructions of microtubules with and without decorating protein-binding partners, but these pipelines can be difficult to implement for the average user, especially for undecorated filaments. Here, we describe MiCSPARC, a cryo-EM processing pipeline developed around CryoSPARC [Punjani et al. (2017), Nat. Methods, 14, 290-296], which leverages automated particle picking and fast 3D refinement times in CryoSPARC to determine the structures of both decorated and undecorated microtubules. We generate reconstructions of undecorated GDP microtubules, as well as kinesin-1 motor domain-decorated GMPCPP filaments, at resolutions of up to 2.8 Å, demonstrating the robustness of the pipeline. Based on its convenient implementation and its ability to routinely generate high-resolution, seam-corrected microtubule reconstructions, MiCSPARC should provide a valuable tool for understanding microtubule dynamics, microtubule-associated proteins and microtubule-targeting agents.
Insights
MiCSPARC is a new cryo-EM pipeline that simplifies the high-resolution structural analysis of microtubules. It enables detailed visualization of both decorated and undecorated microtubule structures.
Area of Science:
- Structural Biology
- Cell Biology
- Biophysics
Background:
- Microtubules are essential cytoskeletal polymers with dynamic structures.
- Existing cryo-electron microscopy (cryo-EM) methods for microtubule reconstruction can be complex, especially for undecorated filaments.
- Variable microtubule lattice architectures pose challenges for structural analysis.
Purpose of the Study:
- To develop an accessible and robust cryo-EM processing pipeline for determining high-resolution microtubule structures.
- To enable the analysis of both decorated and undecorated microtubules using automated cryo-EM workflows.
- To provide a tool for researchers studying microtubule dynamics and interactions.
Main Methods:
- Development of MiCSPARC, a cryo-EM processing pipeline integrated with CryoSPARC.
- Leveraging automated particle picking and rapid 3D refinement within CryoSPARC.
- Application of the pipeline to undecorated GDP microtubules and kinesin-1 decorated GMPCPP filaments.
Main Results:
- Achieved high-resolution cryo-EM reconstructions of microtubules up to 2.8 Å.
- Demonstrated the pipeline's effectiveness for both decorated and undecorated microtubule samples.
- Generated seam-corrected microtubule reconstructions, improving structural accuracy.
Conclusions:
- MiCSPARC offers a user-friendly and efficient approach for high-resolution microtubule structure determination.
- The pipeline facilitates the study of microtubule architecture, dynamics, and interactions with associated proteins.
- MiCSPARC is a valuable tool for advancing research in microtubule-related fields, including drug discovery.
