VitisC: Visualizing small proteins via the integrated scaffold of Escherichia coli CTP synthase (CTPS) cytoophidium

Cryo-electron microscopy (cryo-EM) single-particle analysis is a powerful method for high-resolution structural determination of macromolecules and complexes. Nevertheless, structural characterization of small proteins remains a major challenge due to their limited molecular size. To address this limitation, we develop a novel cryo-EM platform termed Visualizing interested target via integrated scaffold of cytoophidium (VitisC). This approach utilizes the filamentous scaffold of the large macromolecule Escherichia coli CTP synthase (CTPS), organized into cytoophidia, to enable structural visualization of small proteins. By artificially fusing small proteins to this symmetrical and stable CTPS cytoophidium scaffold, we optimize the in vitro conditions for forming stable filamentous complexes. Cryo-EM single-particle analysis of the fusion complex achieve an overall resolution of 3.39 Å, allowing visualization of the small protein density. Furthermore, we establish a general workflow illustrating how VitisC can be adapted for broader applications. This work not only validates the utility of cytoophidia (specifically CTPS-based cytoophidium) as effective scaffolds for cryo-EM imaging, but also provides a generalizable strategy for high-resolution structural analysis of small proteins, expanding the applicability of cryo-EM to previously challenging targets.