Toward the First Structural Characterization of Heterophrynus longicornis Hemocyanin by Cryo-EM: Optimization of a Single-Particle Analysis Workflow

Hemocyanins are large allosteric proteins responsible for oxygen transport in several arthropods and have historically served as important model systems for the development of image-processing methodologies in electron microscopy. Despite advances in structural biology, the structural basis underlying their allosteric properties and cooperative oxygen binding remains poorly understood. This project aims to develop and optimize methodologies in IMAGIC-4D for single-particle analysis (SPA), with emphasis on the assessment of resolution, information content, and structural variability at local and global scales. The hemocyanin from Heterophrynus longicornis (Amblypygi), for which no structural information is currently available, is being investigated as a model system of ecological relevance in Brazil. A hemolymph sample from H. longicornis, obtained in collaboration with the Butantan Institute, was used to establish experimental protocols. Initially, serial dilutions were prepared for negative-stain analysis and evaluated using a JEOL JEM-1400Flash transmission electron microscope, allowing assessment of particle presence, distribution, and sample quality and the identification of preliminary conditions for cryo-EM. Based on this screening, cryo-EM grids were prepared at different sample concentrations to optimize particle distribution and density within the ice. Cryo-EM datasets were subsequently acquired using a Titan Krios microscope equipped with a K3 direct electron detector and are currently being analyzed by SPA. The project has advanced the implementation and optimization of SPA protocols for large macromolecular complexes by integrating sample preparation and assessment, optimization of data-acquisition conditions, and computational processing. Analysis of the acquired datasets will guide the optimization workflow for resolution assessment and structural variability analysis in IMAGIC-4D, supporting the structural characterization of H. longicornis hemocyanin at high resolution and contributing to the investigation of its oligomeric organization and allosteric mechanisms.