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

Alexander D Cook,Szymon W Manka,Su Wang,Carolyn A Moores,Joseph Atherton

doi:10.1016/j.jsb.2019.10.004

Alexander D Cook, Szymon W Manka + Show 3 more

Open Access

https://doi.org/10.1016/j.jsb.2019.10.004

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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.

Highlights

Structure determination using cryo-electron microscopy is a powerful and widely applicable methodology
Several groups have successfully implemented MT “single particle” methods (Sindelar and Downing, 2007; Zhang and Nogales, 2015), thereby solving the structure of pseudo-helical MTs to near-atomic resolution (Kellogg et al, 2018; Manka and Moores, 2018b; Vemu et al, 2017; Vemu et al, 2016; Zhang et al, 2015; Zhang et al, 2018; Zhang et al, 2017), including in the absence of fiducial marker-like binding partners (Zhang et al, 2018). Inspired by these previous studies and by the utility and popularity of RELION, we developed a Microtubule RELION-based Pipeline (MiRP)
As will be described in more detail, the biggest challenges are identifying the Rot angle and translations, they are the focus of MiRP design

Summary

Introduction

Structure determination using cryo-electron microscopy (cryo-EM) is a powerful and widely applicable methodology. Developments in both hardware and software have led to recent radical improvements in attainable resolutions, expansion of the types of samples that can be studied, and in experimental throughput (Kuhlbrandt, 2014). It has been developed to allow new users to determine structures, while allowing more sophisticated interventions by experienced practitioners (Scheres, 2012). It is open-source, actively maintained and updated, and has an engaged and knowledgeable community of users. It’s most recent release (v3.0) incorporated Bayesian polishing and per particle CTF correction (Zivanov et al, 2018)

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