Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development

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.

Keywords: Mycobacterium tuberculosis; cryo-EM; expression and purification protocols; ferritin; single-particle analysis.

Figure 1

Single-particle analysis of BfrB. (a) A micrograph of a highly concentrated (80 mg ml⁻¹) BfrB sample in vitreous ice collected on a Falcon 3 at 200 kV. (b) 2D class averages; the size of the shown box is 150 Å. (c) 3D reconstruction from 163 568 particles at 2.12 Å resolution collected on a K3 at 300 kV. (d) Gold-standard Fourier shell correlation (FSC) before (red line) and after (orange line) masking and the phase-randomized FSC (black line).

Figure 2

Representative regions of the density. (a) Post-processing map density for Phe23 and Tyr35. Local resolution scaled maps of (b) His175, (c) Glu5–Thr9 and (d) C_rigid (Pro174–Leu181). (e) A string of density blobs near the ferroxidase sites. (f) C_flex (Val164–Ala173). The EM density is shown as a grey mesh; the residue atoms are represented as a ball-and-stick model.

Figure 3

Density map and model for C_flex, which extends into the interior of the cage and is located above the cavity between the B-pore and the ferroxidase centres.

Figure 4

Simple purification workflow of the BfrB protein. After protein expression, E. coli cells are lysed by sonication and cellular debris is removed by centrifugation. The protein sample was precipitated by ammonium sulfate and resuspended for further purification by size-exclusion chromatography. A high yield of highly pure protein suitable for cryo-EM studies was obtained.