Abstract
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 Å 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.
Highlights
Single-particle cryogenic electron microscopy has become an indispensable tool for structural biology
As of March 2021, 11 of the 25 single-particle analysis (SPA) structures deposited in the Protein Data Bank (PDB) with a resolution of better than 2 Aare of ferritins from different organisms
bacterioferritin B (BfrB) could serve the structural community in testing their expanding fleet of equipment as well as aiding in a better understanding of iron-storage proteins, which are essential for the survival and the progression of important human pathogens such as M. tuberculosis
Summary
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’ (Kuhlbrandt, 2014), since which an increasing number of single-particle analysis (SPA) structures with resolutions below 2 Ahave been determined (Zivanov et al, 2018; Bartesaghi et al, 2018; Hamaguchi et al, 2019; Tan et al, 2018). BfrB could serve the structural community in testing their expanding fleet of equipment as well as aiding in a better understanding of iron-storage proteins, which are essential for the survival and the progression of important human pathogens such as M. tuberculosis
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