A microtubule RELION-based pipeline for cryo-EM image processing

• Published in: Journal of structural biology Vol. 209; no. 1; p. 107402
• Main Authors: Cook, Alexander D., Manka, Szymon W., Wang, Su, Moores, Carolyn A., Atherton, Joseph
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2020; Academic Press
• Subjects: 3D reconstruction; Cryo-EM; Cryoelectron Microscopy - methods; Databases, Factual; Humans; Image Processing, Computer-Assisted - methods; Microtubule; Microtubule-Associated Proteins - metabolism; Microtubules - chemistry; Microtubules - metabolism; Microtubules - ultrastructure; Pseudo-helical symmetry; RELION; RELION; 3D reconstruction; CTF; PF; MT; Microtubule; Pseudo-helical symmetry; cryo-EM; MiRP; Cryo-EM
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2019.10.004

[Display omitted] •MiRP is a pipeline for processing cryo-EM images of microtubules in RELION.•MiRP manages microtubule heterogeneity and pseudo-symmetry.•MiRP reduces errors in angular and translational alignment.•MiRP improved reconstructions from three different microtubule datasets. Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.