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v3.3.3
Co-organised with TIFR Annual Chemistry Conference (TACC-2025)
Abstract: Structural biology generally requires homogeneous sample preparation, however, the workflow of cryoelectron microscopy allows computational sorting of heterogeneous samples. We are utilizing the single particle reconstruction pathway to sort highly heterogeneous samples. The first example is involves structural analysis of key enzymes and protein complexes required by the malarial parasite Plasmodium falsiparum to invade host erythrocytes. The blood stage of malaria is the most damaging, as Plasmodium parasites multiply in Red Blood Cells (RBCs) which can cause severe complications. Protein complexes secreted/displayed by the parasite were subjected to chromatography and density gradient ultracentrifugation for size based separation. Fractions with substantial protein content were cryofrozen and images captured in near native stage, which resulted in highly heterogeneous populations of particles. The images were subjected to reiterative 2D and 3D classifications and refinement protocols to obtain atomic resolution structures of protein complexes such as proteasomes and aminopeptidase enzyme complexes. The identity of the complexes were determined from the cryoEM density by application of cryoID, a method for bottom-up structural proteomics. We find that the native structures have significant differences compared to recombinant protein complexes and also capture dynamic cellular processes involved in the blood stage of the parasite. CryoEM was also applied to sort out conformationally different virus particles in the process of disassembly and genome release, which resulted in a step-by-step understanding of alteration in virus capsid and encapsidated RNA structures. Structural analysis of native complexes and viral disassembly intermediates are expected to aid in the development of more effective drugs and vaccine candidates.
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