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

J Struct Biol. 2020 Jan 1;209(1):107402. doi: 10.1016/j.jsb.2019.10.004. Epub 2019 Oct 11.

Abstract

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.

Keywords: 3D reconstruction; Cryo-EM; Microtubule; Pseudo-helical symmetry; RELION.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Databases, Factual
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Microtubule-Associated Proteins / metabolism
  • Microtubules* / chemistry
  • Microtubules* / metabolism
  • Microtubules* / ultrastructure

Substances

  • CAMSAP1 protein, human
  • Microtubule-Associated Proteins