research papers Acta Cryst. (2021). D77, 1077–1083 https://doi.org/10.1107/S2059798321007233 1077 Received 2 May 2021 Accepted 13 July 2021 Edited by K. R. Vinothkumar, National Centre for Biological Sciences-TIFR, India ‡ These authors contributed equally. Keywords: cryo-EM; ferritin; Mycobacterium tuberculosis; single-particle analysis; expression and purification protocols. EMDB reference: bacterioferritin B, EMD-12738 PDB reference: bacterioferritin B, 7o6e Supporting information: this article has supporting information at journals.iucr.org/d Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development Abril Gijsbers,‡ Yue Zhang,‡ Ye Gao, Peter J. Peters and Raimond B. G. Ravelli* Maastricht Multimodal Molecular Imaging Institute, Division of Nanoscopy, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. *Correspondence e-mail: rbg.ravelli@maastrichtuniversity.nl The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 A ˚ resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164– 181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows. 1. Introduction Single-particle cryogenic electron microscopy (cryo-EM) has become an indispensable tool for structural biology. The combination of direct electron detectors, motion correction (Scheres, 2014; Li et al., 2013), high-end electron microscopes and advanced imaging-processing algorithms allowed the ‘resolution revolution’ (Ku ¨ hlbrandt, 2014), since which an increasing number of single-particle analysis (SPA) structures with resolutions below 2 A ˚ have been determined (Zivanov et al., 2018; Bartesaghi et al., 2018; Hamaguchi et al., 2019; Tan et al., 2018). Hardware improvements such as monochromators, spherical aberration (C S ) correctors, energy filters and a new generation of direct electron detectors have improved the signal-to-noise ratio even further and have brought SPA to real atomic resolution (Nakane et al. , 2020; Yip et al. , 2020). The success of SPA has attracted many new scientists into the field of cryo-EM and has led to an impressive growth in the community as well as in the number of instruments that can deliver high-resolution cryo-EM structures. Ferritin is often used as a benchmark to commission and validate these machines, as it is relatively straightforward to obtain good- resolution data sets for its 24-mers. In fact, as of March 2021, 11 of the 25 SPA structures deposited in the Protein Data Bank (PDB) with a resolution of better than 2 A ˚ are of ferritins from different organisms. Ferritin has thus become a gold standard for cryo-electron microscopists to evaluate their setups and to push the limits of sample preparation (Ravelli et al., 2020; Russo & Passmore, 2014; Jain et al., 2012; Carragher et al., 2019), imaging (Yip et al. , 2020; Nakane et al. , 2020) and data processing (Zhang et al., 2020; Zivanov et al. , 2018; Punjani et al., 2017). Ferritins are protein complexes that are involved in iron homeostasis and DNA repair by storing iron in cage-like ISSN 2059-7983