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

Area of Science:

• Structural Biology
• Biochemistry
• Microbiology

Background:

• Cryo-electron microscopy (cryo-EM) is expanding globally, necessitating standardized, high-quality protein samples.
• Mycobacterium tuberculosis bacterioferritin B (BfrB) is a potential standard protein for cryo-EM workflows.
• Efficient expression and purification protocols are crucial for advancing structural studies.

Purpose of the Study:

• To develop a straightforward protocol for producing high-yield, high-purity apoferritin, bacterioferritin B (BfrB) from Mycobacterium tuberculosis.
• To determine the high-resolution cryo-EM structure of BfrB.
• To characterize unique structural features of BfrB that may contribute to its stability.

Main Methods:

• Bacterial expression and purification of Mycobacterium tuberculosis BfrB.
• Single-particle cryo-electron microscopy (cryo-EM) data collection and processing.
• 3D reconstruction and model refinement to determine the BfrB structure.

Main Results:

• A simple protocol yielding high-purity BfrB with high efficiency was established.
• A 2.12 Å resolution cryo-EM structure of BfrB was obtained, revealing a 24-mer cage-like oligomer.
• A unique C-terminal extension (residues 164-181) was identified, looping into the protein core and enhancing stability. This region was previously ambiguous in crystal structures.

Conclusions:

• The developed protocol provides a reliable source of BfrB for cryo-EM applications.
• The high-resolution structure and identified C-terminal extension offer insights into BfrB stability and oligomerization.
• This work supports the cryo-EM community by providing a valuable protein standard and structural data.