Dataset Information

Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.

ABSTRACT: Cryogenic electron microscopy (cryo-EM) has recently emerged as an optimal technique for the determination of histone methyltransferase-nucleosome complex structures. Histone methyltransferases are a group of enzymes that posttranslationally methylate histone lysine and arginine residues on the nucleosome, providing important epigenetic signals that regulate gene expression. Here we describe a protocol to solve the structure of histone lysine methyltransferase Dot1L bound to a chemically ubiquitylated nucleosome, including complex reconstitution, crosslinking, grid preparation, and data collection and analysis. Throughout, we discuss key steps requiring optimization to allow this protocol to serve as a starting point for the determination of new histone methyltransferase-nucleosome complex structures.

SUBMITTER: Spangler CJ

PROVIDER: S-EPMC10202144 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.

Spangler Cathy J CJ McGinty Robert K RK

Methods in molecular biology (Clifton, N.J.) 20220101

Cryogenic electron microscopy (cryo-EM) has recently emerged as an optimal technique for the determination of histone methyltransferase-nucleosome complex structures. Histone methyltransferases are a group of enzymes that posttranslationally methylate histone lysine and arginine residues on the nucleosome, providing important epigenetic signals that regulate gene expression. Here we describe a protocol to solve the structure of histone lysine methyltransferase Dot1L bound to a chemically ubiquit ...[more]

PMID: 35733015

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Project description:Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.ImportanceMalaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Dataset Information

Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.

Publications

Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.

Similar Datasets

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