ABSTRACT The intracellular parasite Toxoplasma gondii provides a model for studying structure-function relationships of conserved features of the phylum Apicomplexa. Several apicomplexan parasites are responsible for severe human diseases that persist despite efforts to control them. One limitation is that we lack fundamental understanding of essential parasite processes that could be used to develop new and/or improved therapies. Our work is motivated by the goal of understanding the unique complexity of structural features that form the cytoskeleton of apicomplexan parasites. Among the conserved features is the apical complex consisting of specialized tubulin rich cytoskeletal structure called the conoid, which provides a platform for other motor proteins and conduit for protein secretion. Toxoplasma has one of the most elaborate conoid structures, and while it is reduced in complexity in other apicomplexans, many of the core proteins are conserved. In T. gondii, the apical complex consists of several preconoidal rings, a prominent conoid made up of specialized microtubular conoidal fibers, two long intraconoidal microtubules, and the base formed by the apical polar ring. To date, only a few proteins have been studied functionally, although it is anticipated that conoid is comprised of ~100 proteins. Progress has been limited by the laborious process of identifying and functionally characterizing proteins in conoid, most of which have no orthologs in model systems. The proposed project aims to elevate the state of knowledge of the composition and function of the conoid by identifying all of the major proteins and localizing them in a composite 3-D structure of the organelle. This goal will be achieved through cell fractionation, quantitative mass spectrometry, and high-resolution single-particle cryo-electron microscopy (cryo-EM). We will also functionally characterize a number of new conoid proteins to define their roles in conoid assembly and function. These findings are expected to provide a near-complete atomic model of all the major components of the conoid, define the role of essential components in its assembly and function, and provide a framework for comparative studies of other apicomplexans.