Aligning the Symmetry of the Type III Secretion System Needle Complex

Advances in cryo-EM single-particle analysis have resulted in the routine determination of molecular structures to resolutions rivalling X-ray crystallography. Determining a reconstruction to high resolution requires a homogeneous particle data set; heterogeneity in conformation, occupancy, or even symmetry-mismatched components within a protein complex can present a challenge in data processing and affect the achievable resolution. The bacterial type III secretion system, or injectisome, is a macromolecular nanomachine used by some Gram-negative bacteria to inject effector proteins into a eukaryotic host to aid bacterial survival. The core dual-membrane-spanning needle complex has been the focus of structural study for the last two decades; however, the varied and mismatched internal symmetries of the highly oligomeric constituent components have presented numerous challenges for cryo-EM single-particle data processing. Here we give an overview of the history of cryo-EM studies of the prototypical Salmonella SPI-1 needle complex and discuss the workflow we recently employed in the successful determination of the entire complex.