In situ and in vitro cryo-EM reveal structures of mycobacterial encapsulin assembly intermediates

Area of Science:

• Structural biology
• Microbiology
• Biochemistry

Background:

• Prokaryotes utilize proteinaceous compartments called encapsulins to sequester harmful cellular reactions.
• Encapsulins are prevalent in bacteria, including Mycobacteriaceae, which harbors human pathogens like Mycobacterium tuberculosis and Mycobacterium leprae.
• Previous structural studies focused on fully assembled encapsulin shells, leaving assembly and cargo encapsulation mechanisms poorly understood.

Purpose of the Study:

• To investigate the dynamic assembly process of encapsulins.
• To characterize intermediate states of encapsulin shell assembly and cargo encapsulation.
• To elucidate the mechanism of encapsulin self-assembly.

Main Methods:

• Combined in situ and in vitro structural electron microscopy techniques.
• Utilized cryo-focused ion beam (FIB) lamella preparation and cryo-electron tomography (CET) to visualize encapsulins within Mycobacterium marinum.
• Employed in vitro cryo-electron microscopy (EM) single-particle analysis of Mycobacterium tuberculosis encapsulin.

Main Results:

• Direct visualization of encapsulins in Mycobacterium marinum revealed ribbon-like attachments, shells with and without cargo, and partially assembled structures.
• Three intermediate and one high-resolution (2.3 Å) structure of the Mycobacterium tuberculosis encapsulin shell were obtained.
• Identified dynamic assembly with intermediate states of cargo encapsulation and shell formation.

Conclusions:

• Encapsulins are dynamic assemblies, not static structures.
• A model for encapsulin self-assembly via pairwise monomer addition is proposed based on intermediate structures.
• Understanding these dynamics provides insights into bacterial defense mechanisms and potential therapeutic targets.